High-purity Rebaudioside D and low-calorie soy sauce containing the same

ABSTRACT

The invention provides methods of purifying Rebaudioside D from the  Stevia rebaudiana  Bertoni plant extract along with Rebaudioside A. The methods are useful for producing high purity Rebaudioside D and Rehaudioside A. The invention further provides a low-calorie soy sauce containing the purified Rebaudioside D and a process for making the low-calorie soy sauce containing the purified Rebaudioside D.

RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 12/580,233, filed Oct. 15, 2009 now U.S. Pat. No.8,299,224.

FIELD OF THE INVENTION

The invention relates to a process for isolation and purification ofindividual sweet glycosides from Stevia rebaudiana Bertoni plantextract, and more particularly to isolation and purification ofRebaudioside D from Stevia rebaudiana Bertoni plant extract and furtherto low-calorie soy sauce sweetened with high intensity sweetener.

DESCRIPTION OF THE RELATED ART

Sweeteners are critical ingredients in food supply. The demand ofhealthy low calorie beverages and food products results in theincreasing consumption of sweeteners; thus there is a need to reduce thecalories contributed by sweeteners. This goal can be achieved by usinghigh intensity sweeteners.

High intensity sweeteners possess sweetness level many times exceedingthat of sucrose. They are essentially non-caloric and used widely inmanufacturing of diet and reduced calorie food. Although natural caloricsweetener such as sucrose, fructose, and glucose provide the mostdesirable taste to consumers, they are caloric. High intensitysweeteners do not affect the blood glucose level and provide little orno nutritive value.

However, high intensity sweeteners that generally are used as sucrosesubstitutes possess taste characteristics different than that of sugar,such as sweet taste with different temporal profile, maximal response,flavor profile, mouthfeel, and/or adaptation behavior than that ofsugar. For example, the sweet taste of some high-potency sweeteners isslower in onset and longer in duration than that of sugar and thuschanges the taste balance of a food composition. Because of thesedifferences, usage of high-potency sweetener in replacing such a bulksweetener as sugar in a food or beverage causes an unbalanced temporaland/or flavor profile. If the taste profile of high-potency sweetenerscould be modified to impart desired taste characteristics, it canprovide low calorie beverages and food products with tastecharacteristics more desirable for consumers.

On the other hand, high-potency sweeteners may have some cost andfunctional advantages compared to sugar. The competition among sugar andnon-sugar high-potency sweeteners is tough in soft drinks industry, incountries where their use and production is permitted and also incountries with overvalued sugar prices.

At present high intensity sweeteners are used worldwide. They can be ofboth synthetic and natural origin.

Non-limiting examples of synthetic sweeteners include sucralose,potassium acesulfame, aspartame, alitame, saccharin, neohesperidindihydrochalcone synthetic derivatives, cyclamate, neotame, dulcin,suosan,N—[N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine1-methyl ester,N—[N-[3-(3-hydroxy-4-methoxyphenyl)-3-methylbutyl]-L-α-aspartyl]-L-phenylalanine1-methyl ester,N—[N-[3-(3-methoxy-4-hydroxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine1-methyl ester, salts thereof, and the like, and combination thereof.

Non-limiting examples of natural high intensity sweeteners includeStevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, RebaudiosideE, Rebaudioside F, Steviolbioside, Dulcoside A, Rubusoside, mogrosides,brazzein, neohesperidin dihydrochalcone (NHDC), glycyrrhizic acid andits salts, thaumatin, perillartine, pernandulcin, mukuroziosides,baiyunoside, phlomisoside-I, dimethyl-hexahydrofluorene-dicarboxylicacid, abrusosides, periandrin, carnosiflosides, cyclocarioside,pterocaryosides, polypodoside A, brazilin, hernandulcin, phillodulcin,glycyphyllin, phlorizin, trilobatin, dihydroflavonol,dihydroquercetin-3-acetate, neoastilibin, trans-cinnamaldehyde, monatinand its salts, selligueain A, hematoxylin, monellin, osladin,pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin,curculin, neoculin, chlorogenic acid, cynarin, siamenoside and others.

High intensity sweeteners can be derived from the modification ofnatural high intensity sweeteners, for example, by fermentation,enzymatic treatment, or derivatization.

At present about eleven high intensity sweeteners are used worldwide.These are acesulfame-K, alitame, aspartame, cyclamate, glycyrrhizin,NHDC, saccharin, Stevioside, sucralose, thaumatin, neotame, andRebaudioside A.

The high intensity sweeteners can be grouped into three generations. Thefirst generation represented by cyclamate, glycyrrhizin and saccharinhas a long history of use in food. The second generation includesacesulfame-K, aspartame, NHDC and thaumatin. Alitame, neotame,sucralose, Stevioside, and Rebaudioside A belong to the thirdgeneration.

The standard sweetening power associated with each high intensitysweetener is given in TABLE 1. However, when they are used in blends,the sweetening power can change significantly.

TABLE 1 Sweetener Sweetness power Saccharose 1 Acesulfame-K 200 Alitame2000 Aspartame 200 Cyclamate 30 Glycyrrhizin 50 NHDC 1000 Saccharine 300Stevioside 200 Rebaudioside A 450 Thaumatin 3000 Sucralose 600

On the other hand, ‘natural’ and ‘organic’ foods and beverages havebecome the “hottest area” in the food industry. The combination ofconsumers' desire, advances in food technology, new studies linking dietto disease and disease prevention has created an unprecedentedopportunity to address public health through diet and lifestyle.

A growing number of consumers perceive the ability to control theirhealth by enhancing their current health and/or hedging against futurediseases. This creates a demand for food products with enhancedcharacteristics and associated health benefits, specifically a food andconsumer market trend towards “whole health solutions” lifestyle. Theterm “natural” is highly emotive in the world of sweeteners and has beenidentified as one of key trust, along with “whole grains”,“heart-healthy” and “low-sodium”. ‘Natural’ term is closely related to‘healthier’.

In this respect, natural high intensity sweeteners can have bettercommercial potential.

Natural caloric sugars, such as sucrose, fructose, and glucose areutilized heavily in beverage, food, dental, and oral hygienic/cosmeticindustries due to their pleasant taste. In particular, sucrose imparts adesirable taste for consumers. Although sucrose provides superiorsweetness characteristics, it is caloric. While calories are necessaryfor proper bodily functions, there is a need in the market to providealternative non-caloric or low-caloric sweeteners with sugar-like tastefor consumers with sedentary lifestyles or those who are calorieconscious. New formulations of beverage, food and dressings productswith zero or low content of sugar are desirable. However, in general,non-caloric or low caloric sweeteners have associated undesirable tastesto consumers such as delayed sweetness onset; lingering sweetaftertaste; bitter taste; metallic taste; astringent taste; coolingtaste; licorice-like taste; and/or the like. If the taste profile ofnatural and synthetic high-potency sweeteners could be modified toimpart specific desired taste characteristics to be more sugar-like, thetype and variety of compositions that may be prepared with thatsweetener would be significantly expanded. Accordingly, it would bedesirable to selectively modify the taste characteristics of natural andsynthetic high-potency sweeteners. It is also very important thatdiabetic (with no sugar) or reduced calorie products have as fewdifferences as possible from conventional items.

The development of new formulations, for example, employing sweeteners,flavorants, flavor enhancing agents and the like, presents challenges inaddressing associated bitterness and/or other off-tastes. In addition,such challenges typically are presented in formulations developed forimproved characteristics and/or flavor profiles. Also, there is need fornew formulations which can satisfactorily meet the combination ofobjectives including nutritional, flavor, shelf life, and otherobjectives.

An object of the invention is to provide a fermented food composition,particularly, low calorie soya sauce, having excellent taste profile,mouthfeel and physical characteristics. The product comprises at leastone non-nutritive natural sweetener in an amount sufficient to provideperceptible sweetening. The composition provides a more sugar-likesweetness profile due to use the new natural high intensity sweetener.

Stevia rebaudiana Bertoni is a perennial shrub of the Asteraceae(Compositae) family native to certain regions of South America. Theleaves of the plant contain from 10 to 20% of diterpene glycosides,which are around 150 to 450 times sweeter than sugar. The leaves havebeen traditionally used for hundreds of years in Paraguay and Brazil tosweeten local teas and medicines.

At present there are more than 230 Stevia species with significantsweetening properties. The plant has been successfully grown under awide range of conditions from its native subtropics to the cold northernlatitudes.

Steviol glycosides have zero calories and can be used wherever sugar isused. They are ideal for diabetic and low calorie diets. In addition,the sweet steviol glycosides possess functional and sensory propertiessuperior to those of many high potency sweeteners.

The extract of Stevia rebaudiana plant contains a mixture of differentsweet diterpene glycosides, which have a single base—steviol and differby the presence of carbohydrate residues at positions C13 and C19. Theseglycosides accumulate in Stevia leaves and compose approximately 10%-20%of the total dry weight. Typically, on a dry weight basis, the fourmajor glycosides found in the leaves of Stevia are Dulcoside A (0.3%),Rebaudioside C (0.6%), Rebaudioside A (3.8%) and Stevioside (9.1%).Other glycosides identified in Stevia extract include Rebaudioside B, C,D, E, and F, Steviolbioside and Rubusoside (FIG. 1). Among steviolglycosides only Stevioside and Rebaudioside A are available incommercial scale.

The chemical structures of the diterpene glycosides of Stevia rebaudianaBertoni are presented in FIG. 2.

The physical and sensory properties are well studied only for Steviosideand Rebaudioside A. The sweetness potency of Stevioside is around 210times higher than sucrose, Rebaudioside A in between 200 and 400 times,and Rebaudioside C and Dulcoside A around 30 times. Rebaudioside A isconsidered to have most favorable sensory attributes of the four majorsteviol glycosides (TABLE 2).

The glycosides from leaves can be extracted using either water ororganic solvent extraction. Supercritical fluid extraction and steamdistillation were described as well. Methods for recovery of diterpenesweet glycosides from Stevia rebaudiana using membrane technology, andwater or organic solvents, such as methanol and ethanol also aredescribed.

TABLE 2 Optical rotation [α]²⁵ _(D) Solubility T_(Melt), Mol. (H₂O, inwater, Relative Quality of Name Formula ° C. Weight 1%, w/v) % sweetnesstaste Steviol C₂₀H₃₀O₃ 212- 318.45 ND ND ND Very bitter 213Steviolmonoside C₂₆H₄₀O₈ ND 480.58 ND ND ND ND Stevioside C₃₈H₆₀O₁₈ 196-804.88 −39.3 0.13 210 Bitter 198 Rebaudioside A C₄₄H₇₀O₂₃ 242- 967.01−20.8 0.80 200-400 Less Bitter 244 Rebaudioside B C₃₈H₆₀O₁₈ 193- 804.88−45.4 0.10 150 Bitter 195 Rebaudioside C C₄₄H₇₀O₂₂ 215- 951.01 −29.90.21 30 Bitter 217 Rebaudioside D C₅₀H₈₀O₂₈ 248- 1129.15 −29.5 1.00 220Like sucrose 249 (ethanol) Rebaudioside E C₄₄H₇₀O₂₃ 205- 967.01 −34.21.70 170 Like sucrose 207 Rebaudioside F C₄₃H₆₈O₂₂ ND 936.99 −25.5 ND(methanol) Dulcoside A C₃₈H₆₀O₁₇ 193- 788.87 −50.2 0.58 30 Very bitter195 Steviolbioside C₃₂H₅₀O₁₃ 188- 642.73 −34.5 0.03 90 Unpleasant 192Rubusoside C₃₂H₅₀O₁₃ ND 642.73 642.73 ND 110 Very bitter

There are several publications on purification of some individualsteviol glycosides.

Generally production of extract includes extraction of plant materialwith water or water-organic solvent mixture, precipitation of highmolecular weight substances, deionization, and decolorization,purification on specific macroporous polymeric adsorbents, concentrationand drying.

U.S. Pat. No. 3,723,410 discloses an extraction of Steviosides fromStevia rebaudiana Bertoni. The method included defatting of Stevialeaves by treatment with chloroform for more than 150 hours at boilingtemperatures and three times treatment with dioxane in the presence ofcalcium carbonate for two hours at boiling temperatures. Afterfiltration the dioxane filtrates were combined and concentrated to syrupstate under reduced pressure at 50° C. An equal volume of methanol wasthen added to the syrup and the resulting solution set aside over nightto allow crystallization to occur. The crystals were collected byfiltration and washed thoroughly with ice cold methanol. The residualsolution was concentrated, an equal volume of methanol was added, andthe mixture set aside overnight to crystallize. The crystals wereremoved by filtration and dried in vacuum at 100° C. The yield ofStevioside was 6.5% from air-dried leaves. The method is verycomplicated with the usage of toxic organic solvents. There is noinformation about purity of Stevioside, however in described conditionsRebaudiosides will precipitate along with Stevioside. The process isdifficult to apply on commercial scale.

A method for the production of Stevia extract with further isolation ofRebaudioside A is developed in U.S. Pat. No. 4,082,858. The air-driedStevia leaves were extracted with hot water, and the extract was driedunder vacuum. The resulted mixture was extracted with methanol and fromcombined extracts methanol was removed by distillation under reducedpressure. The obtained syrup was subjected to chromatographic separationon a silica gel column using mixture of n-propanol, water and ethylacetate as mobile phase. The method is useful in laboratory scale onlyand has various disadvantages on the commercial scale.

U.S. Pat. No. 4,171,430 discloses a purification of Stevioside fromStevia extract. The method included extracting Stevia leaves with water,concentrating the solution and extracting with methanol. Stevioside wascrystallized from methanol solution and purified on styrene type gelwith tetrahydrofuran as mobile phase. The method is useful in laboratoryscale only. The process is difficult to apply on commercial scale.

U.S. Pat. No. 4,361,697 discloses an extraction, separation and recoveryof diterpene glycosides from Stevia rebaudiana. The process included thesteps of sequential extracting of plant material first with a solvent ofintermediate polarity (such as chloroform), and then with a secondsolvent of high polarity (such as methanol). The resulting extract wassubjected to a liquid chromatography separation. The steviol glycosideswere in the methanol fraction. The major drawbacks were the use ofvarious toxic solvents to extract and process sweet glycosides. Finalpurification of glycosides was achieved by column chromatography usingsorbents like silica gel as a stationary phase and eluting the columnwith two solvents sequentially running through the column. Process isnot environmentally-friendly and difficult to carry out in the largescale.

An improved method for the recovery of steviol glycosides from Steviarebaudiana Bertoni plant, which does not require the use of specialseparation equipment such as ion exchange and/or chromatographic columnswas described in U.S. Pat. No. 4,599,403. The extraction was carried outwith water. The resulting aqueous extract is treated with citric acid toremove metallic and other impurities as well as to lower the pH to about3.0. The mixture was filtered through Celite and pH of the filtrateadjusted to 10.5 by calcium oxide. The formed precipitate was remover byfiltration. The filtrate was concentrated and extracted with n-butanol.Purified Stevioside crystals were then recovered by cooling the waterlayer obtained from the solvent extraction step. The major drawbacks ofthe method are the losses of glycosides during extraction by n-butanoland also low yield of Stevioside crystals from aqueous solution. Thesalt content in the final product can be high. There are no data aboutthe final purity of Stevioside. The process is difficult to apply oncommercial scale.

U.S. Pat. No. 4,892,938 and JP No. 01-131191 disclose a purificationprocess in which the extract of the plant was obtained through treatmentin water at a temperature from room to about 65° C. with stirring andsubsequent filtration and centrifugation. This extract was treated withcalcium hydroxide and the precipitate was removed by filtration orcentrifugation. This filtrate was treated with a strong acidic ionexchange resin and subsequently with a weak basic ion exchange resin.The sweet glycosides remained in the water and were recovered byevaporation of the water. The disadvantage is that the final product hasquite low purity. The sweet glycosides content in the final product wasonly about 70%.

U.S. Pat. No. 5,112,610 discloses a natural sweetener preparationprocess based on Stevia rebaudiana. The method included extracting theplant material of Stevia rebaudiana with an organic solvent andsubjecting the solution to supercritical gas (CO₂) extraction to obtaina residue, which was free from undesired and taste-impairingconstituents. Generally speaking, the method concerned to removal ofcurticle waxes, chlorophyll, other pigments and especiallytaste-impairing components from Stevia leaves or extract. However directtreatment of the leaves required a great quantity of starting materialso that the use of leaves was non-economical even when increasing thebulk density of the dried or comminuted leaves by pressing into pelletsprior to the extraction. The treatment of powdered extract, which wasobtained from leaves by conventional method, allowed the removal oftaste-impairing components only to a lesser degree, and withoutemploying entrainers (low molecular weight alcohols, suitablehydrocarbon or mixture of the solvents) achieves not entirelysatisfactory results. Moreover, there are no quantified data on theactual purity of extract. The process is difficult to apply oncommercial scale.

U.S. Pat. No. 5,962,678 describes a multi-step extraction andpurification process of Rebaudioside A from Stevia rebaudiana plant. Theextract of the plant was obtained through treatment in water at atemperature ranging from ambient to about 65° C. with stirring andsubsequent filtration and centrifugation. This extract was treated withcalcium hydroxide and the precipitate was removed by filtration orcentrifugation. This filtrate was treated with a strong acidic ionexchange resin and subsequently with a weakly basic ion exchange resin.The sweet glycosides remained in the water and were recovered byevaporation of the water. The content of steviol glycosides in theextract in this stage was 70% only. For further purification the productwas passed through the column with Amberlite XAD-7, which was able toadsorb steviol glycosides. After washing with water the glycosides weredesorbed with methanol. The purity of the extract was around 95% withcontent of significant amount of so called yellow oil. To isolateindividual Stevioside and Rebaudioside A the dried solid was refluxed inanhydrous methanol solution and then cooled to precipitate Steviosidewith 91.6% of purity. However, the yield of Stevioside was only 15% fromthe Stevia extract containing 60% Stevioside. Stevioside can be furtherpurified by refluxing it in methanol-water solution. Purity of theproduct was about 99%.

A more purified product can be produced by the combined use ofmicrofiltration, ultrafiltration, and nanofiltration as it is describedin U.S. Pat. No. 5,972,120. The extraction was uninterruptedly carriedout in continuous flow columns. The optimum mean particle size of leaveshad to be about 20 mm. With smaller particles, the filtration ratesubstantially decreased as the column was blocked. Initial water wasadded in a quantity of 0.05 parts per one part of dry leaves (byweight). The column temperature was set to not more than 4° C., andextraction was carried out with water at pH within the range 2.0-4.0(adjusted with phosphoric acid). At low temperatures and pH, a moreselective extraction occurred and nearly colorless solution wasobtained. The extract was then filtered through tubular ceramicmembranes and, then, through ultrafiltration membranes. The producedfiltrate was separated from low-molecular impurities on nanomembranes atelevated temperatures.

Method of preparation of Stevia extract is described in U.S. Pat. Nos.6,031,157 and 6,080,561. The dry leaves were extracted with 10 to 20parts of water several times. The resulting extracts were combined andpassed slowly through a column filled with cation-exchange resin andthen a column filled with anion-exchange resin. The treated solutionthen was passed through a column packed with a resin (Amberlite XAD-2)to adsorb the sweetening components, and then washed with water. Afterthe water was drained from the column, it was eluted with three volumesof methanol to isolate the sweetening components. The effluent wasconcentrated and further dried under a reduced pressure to obtain a paleyellow powder. The major drawback of the method is the low quality ofextract. Treatment with ion-exchangers and specific adsorbents only,cannot result in high quality Stevia extract with white color and highcontent of steviol glycosides.

U.S. Published Patent Application No 2006/0142555 discloses a processfor the production of Steviosides from Stevia rebaudiana plant. Themethod included extraction of plant powder by direct steam injectioninto the extractor followed by filtration to get aqueous extract andcalcium hydroxide treatment to remove impurities in the form ofprecipitate. The filtrate was treated with strong cation-exchange resinand then weak base anion-exchange resin. The aqueous eluate containingSteviosides was concentrated to obtain purified Steviosides with45.47-65.5% Stevioside content in the final product. The provided methodis suitable for production of Stevia extract with various content ofStevioside but not for highly purified steviol glycosides.

U.S. Patent Application Publication No. 2006/0083838 reports a method ofisolating and purifying Rebaudioside A from commercially availableStevia rebaudiana starting material. The method comprised: (1) an EtOHformulation stage to formulate a selected EtOH solvent, (2) a firstreflux stage using the Stevia starting material and optionallyadditional reflux stages using retentate isolated from a refluxedmixture or a stirred wash mixture, (3) optionally, one or more stirredwash stages, and (4) an ethanol purge and drying stage. In embodimentsthat used lower quality Stevia starting material, a second reflux stagewas typically added before the stirred wash stage to maximize purity ofthe Rebaudioside A final product. In the reported method, an EtOHformulation stage was conducted in order to formulate a desired refluxsolvent for use in the reflux step(s). Typically, the reflux solvent wasa mixture of ethanol and water with about 5% to 15% by volume water. Theprocess further included one or more energy-intensive refluxing stepsthat were typically conducted at a temperature of about 89° C. to 90° C.for about 1 hour. The method reportedly produced 100% pure,water-soluble Rebaudioside A.

U.S. Patent Application No. 2006/0134292 reports a process forrecovering sweet glycosides from Stevia rebaudiana plant material. Thedried and powdered leaves were treated with water in the presence of apectinase, cellulase, and alpha-amylase. The use of such enzymes wasreported to considerably increase the extraction rate and facilitatesthe next stages of purification. The resulting extract was purifiedusing treatment with calcium hydroxide and ultrafiltration. Permeate waspassed through the column packed with bentonite and concentrated tosyrup state under vacuum. The treatment with ethanol allowed separatingthe practically pure Rebaudioside A from the mixture. The Rebaudioside Awith high purity was obtained after washing the crystals with 88-95% ofethanol.

U.S. Patent Application No. 2007/0082103 reports a process for preparingof Stevia extract and highly purified Stevioside and Rebaudioside A. Thedried and powdered leaves were subjected to water extraction and theresulted extract was purified using treatment with a base such ascalcium hydroxide and then iron chloride. The filtrate was deionizedusing ion-exchange resins, concentrated under vacuum and spray dried.Highly purified Rebaudioside A and Stevioside were obtained bydissolving the extract in methanol to precipitate Stevioside. Theremaining solution after isolation of Stevioside was dried andRebaudioside A was isolated by treatment with ethanol. The finalpurification of Rebaudioside A was developed by treatment withethanol-water solution. The purity was at least 98%.

U.S. Published Patent Application No 20070292582 discloses purificationof Rebaudioside A. The method comprised the steps of combining crudeRebaudioside A and an aqueous organic solvent to form a Rebaudioside Asolution, the aqueous organic solution comprising water in an amountfrom about 10% to about 25% by weight, and crystallizing from the crudeRebaudioside A solution, in a single step, substantially pureRebaudioside A in purity greater than 95%. In the case ofethanol-methanol-water mixture the yield of Rebaudioside A with puritymore than 97% was 32.5% from starting material containing 77.4%Rebaudioside A. The yield from starting material containing 80.37%Rebaudioside A was in the range of 54.6-72.0%. Other co-solvents usedalong with ethanol such as ethyl acetate, 1-butanol, 2-butanol,tert-butanol, sec-butanol, acetonitrile, isopropanol, and 1-propanolwere not suitable for the production of Rebaudioside A with greater than97% purity. In the case of use ethanol with various amounts of water ascrystallization solvent the yield of Rebaudioside A was in the range39.6%-76.4% from starting material containing 80.37% Rebaudioside A. Theprocess used the mixture of two organic solvents, which recovery andpurification in large scale was very complicated. Moreover, incommercial scale when centrifugation may take relatively long time, theco-precipitation of Stevioside, Rebaudioside C, and Rebaudioside D mayoccur.

U.S. Patent Application No. 2008/0300402 and Chinese Patent No 101200480report a method for producing purified Rebaudioside A comprising thefollowing steps: separation of Rebaudioside A on chromatographic columnpacked with silica gel using the mixture of ethyl acetate, ethanol andwater as mobile phase. Rebaudioside A fractions were combined and dried.The solid was treated with ethanol containing from 2 to 10% of water andRebaudioside A was crystallized by cooling the mixture at −20° C. Thepurity of Rebaudioside A can reach to more than 99%. For thepurification of Rebaudioside A the filtrate after separation ofStevioside was concentrated and cooled to 0° C. overnight for about 16hours. The resulting precipitate of Rebaudioside A was filtered, washedwith a small volume of cold methanol, and dried to obtain Rebaudioside Awith 79.0% purity and 3.3% yield from initial extract. This crudeRebaudioside A was further purified by refluxing in anhydrous methanolor methanol-water mixture. From starting material containing 90.2% ofRebaudioside A the output of the product was around 67% with 98.6% ofpurity. However the method of improving the purity of Rebaudioside Afrom 79% to 90.2% is not available. The major drawback of the process islow yields of the final highly products, which makes the process notsuitable for commercial production of highly purified Stevioside andRebaudioside A.

Various Japanese patents also concern about the preparation of extractfrom Stevia rebaudiana Bertoni.

JP No. 52-100500 describes the purification and concentration of aqueousStevioside extract by treating the extract with specific ion-exchangeresin of high decolorizing capacity, followed by treatment withAmberlite XAD type specific adsorbent. Treatment with onlyion-exchangers and adsorption/desorption is unable to result in highquality extract.

JP No. 52-136200 discloses a preparation of Stevioside solution byextraction with hot water or hydrous alcohol followed by membraneseparation. The molecular weights of sweet glycosides and sterebins arevery close and membrane systems cannot result satisfactory resolution ofthese compounds, which will affect to the purity of extract. Content ofsalts in the final product will be high.

JP No. 52-005800 discloses a method of preparation of purifiedStevioside from leaves of Stevia rebaudiana by extraction and treatmentwith cation-exchange resin. Such treatment will result in yellow powderwith apparently low content of sweet glycosides.

Japanese Patent JP54030199 discloses the process for preparation ofStevia sweetening agent free from characteristic smell and bitter taste,by extracting leaves of Stevia rebaudiana Bertoni with water, treatingthe extract with a non-polar synthetic adsorbent resin followed bydesorption, and further treating with an ion-exchange resin. The processis very similar to traditional Chinese technology, which allowsproducing Stevia extract with steviol glycosides content not more than85-86%.

JP No. 54-132599 discloses a separation and purification of Steviosideby extracting Stevia leaves with hot water, treating the extract with anon-polar synthetic adsorbent, washing the resin with an aqueoussolution of slaked lime, and eluting the Stevioside from the resin witha hydrophilic organic solvent or hydrous hydrophilic organic solvent.Treatment with only non-polar synthetic adsorbent is unable to result inhigh quality extract; no measures are taken for residual salts and thecolor of the product.

JP No. 55-159770 concerns the extraction and purification of Steviosideby extracting Stevia leaves with water or hydrous alcohol. The extractwas concentrated to solid content from 10 to 50%, added 0.1-5.0% ofcalcium chloride to coagulate and precipitate the colloidal impuritiesexisting in the extract. From concentrated solution using CaCl₂ most ofimpurities cannot be removed. There are no desalting and decolorizingstages.

JP No. 55-162953 concerns the preparation of Stevioside by extractingStevia leaves with 10-15 volumes of water at 60-80° C. The extract wastreated with slaked lime with aeration, and the pH of suspension wasadjusted to around 8.0 by adding sulfuric or citric acid. The resultingslightly soluble salt was filtered off and the filtrate was thencontacted with a polyamide resin to remove impurities. The filtrate wasfurther extracted with n-butanol and the organic phase was distilledunder the vacuum to recover the Stevioside as white crystals. Content ofsalts in such product will be high. Purification process using then-butanol extraction is difficult to apply on commercial scale.

JP No. 55-081567 describes the extraction and purification ofStevioside. The extract of Stevia leaves prepared by water or hydrousalcohol extraction was concentrated, and one or more types ofwater-soluble salts of Ca, Fe, and Al and a water soluble organicsolvent, e.g. ethanol or acetone, were added to the concentrate toprecipitate and remove the colloidal impurities. The resulting liquidwith pH 3-7 was passed through a strong cation-exchange resin and a weakanion-exchange resin. The obtained solution was passed through thespecific adsorbent. The fractions of Stevioside were combined. Theprocess is similar to the traditional Chinese technology, which canresult in yellow powder with only 85-86% steviol glycosides content.

JP No. 55-120770 concerns the purification of Stevioside solution. Theleaves and stalks of Stevia rebaudiana Bertoni were extracted with wateror an alcoholic solution, to which a water-soluble tin salt, e.g.,stannous chloride, stannous sulfate, stannic sulfate, etc, was added anddissolved. An alkali substance, e.g., sodium hydroxide or lime was addedto the resulting solution to adjust the pH value around 5-10. The formedprecipitate was separated. This process is unable to result in extractfree from salts and other low-molecular weight impurities.

JP No. 55-138372 describes the purification of Stevioside solution.Stevioside was extracted from the leaves and stalks of Stevia rebaudianawith water, hot water, or a hydrous alcohol, and the extract or itsconcentrate was mixed with slaked lime or lime milk. The mixture wasthen filtered and mixed with an equimolar amount of water-soluble ironcompound, e.g. ferrous sulfate, and stirred to precipitate the iron ionsas a sparingly soluble hydroxide, which was removed with the coloringsubstances adsorbed on it. The process is unable to result in extractfree from salts and other low-molecular weight impurities.

JP No. 55-039731 concerns the extraction of Stevioside. 1 kg of driedleaves of Stevia rebaudiana was extracted with 3-10 volumes of water orhydrous alcohol. The extract was concentrated to solid content of 10-50%and 0.1-5% of a metallic chloride, e.g. calcium, aluminum, or ironchloride, was added. The precipitate of impurities was removed byfiltration. The subsequent purification procedures with ion-exchangeresins, adsorbent, and ultrafiltration membranes can be carried outfurther. Most of impurities cannot be removed from concentrated solutionusing salts. The content of low-molecular weight impurities can be high.

JP No. 56-160962 discloses a purification of Stevioside containingsolution by extracting Stevia leaves with water, concentrating theextract obtained to 25-50% solids content, mixing the concentrate with alow molecular weight aliphatic alcohol, and removing the precipitatedimpurities from the mixture. The amount of the alcohol was at least 5times volume of the aqueous extract, or 3-6 times volume of theconcentrate. The treatment is not suitable to remove low-molecularweight impurities. There are no decolorizing stages. Process isdifficult to apply on commercial scale.

JP No. 56-109568 discloses a purification of Stevia sweeteningsubstances by extracting Stevia leaves with water or hydrophilic organicsolvent. The extract was treated with an organic solvent selected fromthe group consisting of 4-8C ether, 4-7C ester, and 1-4C organicchlorine compound, and the ingredient soluble in the solvent wasseparated. Diethyl ether, diisopropyl ether, ethyl acetate, methylchloride, carbon tetrachloride, etc. may be cited as the purifyingsolvent. The bitter taste can be removed effectively with simultaneousdecolorizing. However, used hazardous solvents, can remain in the finalproduct. Process is difficult to apply on commercial scale.

JP No. 56-099768 concerns the preparation of steviol glycosides. Asolution containing steviol glycosides, e.g. an aqueous extract ofStevia rebaudiana Bertoni, was treated with magnesium silicate aluminateto adsorb impurities, e.g. pigments or proteins. However, salts contentin the final product can be high. There are no decolorizing andadditional purification stages. Steviol glycosides content in the finalproduct can be low.

JP No. 57-002656 concerns the discoloration and purification of Steviaextract. Stevia extract was treated with an aqueous solution of a bariumcompound that is readily soluble in water and then neutralized withsulfuric acid. Barium hydroxide was added until pH was 7-9 and thesuspension again was treated with sulfuric acid to pH 3-4. Theprecipitate was separated. The main drawbacks are that salt content inthe final product can be high, there are no decolorizing and additionalpurification stages, and, as a result, steviol glycosides content in thefinal product can be low.

JP No. 57-005663 concerns the purification of Stevioside throughextraction. An extracted solution of Stevia leaves with water orwater-containing alcohol was concentrated to 10-50% of solids content. Asalt or a base of calcium, iron, or aluminum was added and theprecipitate was removed by filtration. The filtrate pH was adjustedbetween 5-7, and the formed precipitate was removed. The filtrate istreated with a cation exchange and an anion exchange resins andevaporated to dryness. The major drawback of the method is the lowquality of extract. The treatment with alkali and ion-exchangers only isnot enough to produce the Stevia extract with white color and highcontent of steviol glycosides.

JP No. 57-046998 concerns the preparation of Stevioside. Raw leaves ofStevia rebaudiana were extracted with 10-20 volumes of water and thefiltrate was treated with calcium hydroxide in an amount of 10-30% ofthe raw leaves weight. The pH of the suspension was then adjusted to 4-6with sulfuric acid or citric acid. After filtration the extract waspassed through a polyamide column to absorb glycosides and removeimpurities. The purified extract was then concentrated under reducedpressure, pII adjusted to 8-9 with aqueous ammonia and extracted withn-butanol to afford crude Stevioside, which was then recrystallized frommethanol. However, the content of residual salts can be high; there isno decolorizing stage; extraction with n-butanol and recrystallizationfrom methanol is not viable commercially.

JP No. 57-075992 concerns the purification of Stevioside. The waterextract of Stevia rebaudiana Bertoni was mixed with a flocculant (e.g.aluminum or polyaluminum chloride) to flocculate and remove thecolloidal impurities, and then treated with a non-polar resin (e.g.Duolite ES-861) to adsorb the sweetening substance. The adsorbedsubstances were eluted with an organic solvent (e.g. methanol, acetone,etc.), and the solution was discolored and purified with activatedcharcoal and activated clay. Activated charcoal can absorb theStevioside firmly from aqueous solution and the decolorizing andpurification effects of activated charcoal can be promoted by thecombined use with activated clay. However, hazardous solvents are used,which can present in the final product. Process is difficult to apply oncommercial scale.

JP No. 57-086264 concerns the isolation of principal sweeteningcomponent of Stevia. Dried stalks and leaves of Stevia were extractedwith cold water, hot water, hydrous alcohol, etc. The extract wascoagulated or precipitated with an adsorbent, and the precipitate wasremoved by filtration or centrifugation to obtain a clear liquidcontaining the sweetening components. The components were adsorbed to asynthetic polymer adsorbent, purified to 80-90% purity, concentrated,dried, and dissolved in 3-8 volumes of hot methanol or hot ethanol.Stevioside and Rebaudioside A were crystallized from the solutionsimultaneously. After complete removing of the solvent, the mixedcrystals were heated together with a 3-6 volumes of alcohol andseparated into the solution part and the solid part by hot filtration.Stevioside can be obtained from the solution and the Rebaudioside A canbe prepared by washing and drying the solid part. Method can result tothe purified Stevioside and Rebaudioside A; however the quality ofextract can be low because of the absence of deionization anddecolorizing stages. The content of low-molecular weight impurities canbe high.

JP No. 58-212759 and No. 58-212760 described the purification of Steviasweetening substance. The leaves of Stevia rebaudiana Bertoni wereextracted with water or an alcohol at pH 4. The extract was treated withcalcium hydroxide and formed precipitate was filtered off. Awater-soluble organic solvent such as methanol was added to thefiltrate, and precipitate was removed. The amount of the water-solubleorganic solvent was from 5% to 50% based on the filtrate. The filtrateobtained was purified by ion-exchange resins or adsorption resin. Themain drawback is that hazardous solvent is used, which can present inthe final product. Process is difficult to apply on commercial scale.

JP No. 58-028246 described the preparation of Stevioside. The raw leafof Stevia was extracted with water, hot water or a water-alcoholmixture, and if necessary the extract was then concentrated. A mixtureof calcium hydroxide with calcium chloride in an amount of 0.5-2.0 timesthat of the solid content in the extract were added to the extract orconcentrated extract preferably while blowing gaseous carbon dioxide.The impurities were precipitated in the form of a colloidal material,which was separated by filtration. However, the extract quality can below because of high content of salts and low-molecular weight compounds.

JP No. 58-028247 concerns the purification method of Steviosidesolution. The raw leaves of Stevia were extracted with water, hot wateror a water-alcohol mixture, and the extract was concentrated. Calciumhydroxide and a water-soluble high polymeric flocculant, e.g.polyacrylamide high polymer, in an amount of 1-2.5 times that of thesolid content in the extract were added to the extract or concentratedextract to precipitate impurities, which were then filtered off. Atransparent and almost colorless Stevioside solution was obtained.However, the extract quality can be low because of high content of saltsand low-molecular weight compounds.

JP No. 59-045848 concerns the preparation of Stevia sweetener with highcontent of Rebaudioside A. Dried leaves of Stevia variety containing1.57 times more Rebaudioside A than Stevioside were extracted with wateror a water-containing solvent. The prepared extracted solution wastreated with a cation-exchange resin and an anion-exchange resin. Thesolution was adsorbed on an absorption resin, eluted with a hydrophilicsolvent, and the solution was concentrated to yield a natural sweetener.The process is similar to the traditional Chinese technology, which canresult to the yellow powder with steviol glycosides content up to 85-86%only.

JP No. 62-166861 concerns the extraction and purification of sweetenercomponent from dry leaves of Stevia. Dried leaves of Stevia rebaudianaBertoni were extracted with 7-14 volumes of water at 50-70° C. for 3-6hr with agitation to obtain a brown liquid extract with total solidscontent of 2-3% and containing 0.7-0.8% of Stevioside. The extract wasconcentrated 7-8 times at about 60° C. under reduced pressure. Theconcentrated liquid was treated with 0.5-2% CaCl₂ to separate impuritiesas flocculent precipitate. The solution was treated with an Al, Mg oxideof an amount corresponding to 15-20% of the solid content at 40-55° C.under vigorous agitation. Then the precipitate was removed byfiltration. The Stevioside can be further purified on specificadsorbents. However, the process is difficult to commercialize; saltsquantity used for the purification of extract is high and there are nodeionization and decolorizing stages. The content of low-molecularweight compounds can be high.

JP No. 06-007108 concerns the method for extracting and separating sweetsubstances of Stevia rebaudiana Bertoni. Leaves of Stevia rebaudianaBertoni were extracted with a water-miscible alcohol such as methanol.The extracted solution was mixed with water and passed through anultrafiltration membranes having 20-150 kDa cutting capacity and thenthrough the ultrafiltration membranes with 1-10 kDa cutting capacity.However, hazardous solvents are used, which can present in the finalproduct. Process is difficult to apply on commercial scale.

JP No. 52083731 deals on isolation and purification of Rebaudioside Aand Rebaudioside B by column chromatography on silica gel. Furtherpurification is developed by crystallization from organic solvents suchas methanol or ethanol.

JP No. 55-092400 concerns the preparation of Stevioside. An aqueoussolution containing Stevioside was extracted with 1H, 1H,5H-octafluoro-1-pentanol. After separating the solvent was distilledoff, and the residue was dried. The precipitate was recrystallized frommethanol. The purity of Stevioside was more than 95%.

JP No. 56-121453, JP No. 56-121454, and JP No. 56-121455 concern theseparation of Stevioside and Rebaudioside A. A mixture of Stevioside andRebaudioside A extracted from the leaves of Stevia rebaudiana Bertoniwas mixed with 75% aqueous solution of methanol and maintained atambient temperature for about 3 hours. The resulted crystals with 65%Stevioside and 25.2% Rebaudioside A content were separated by filtrationand dried. In the case of application of 90% aqueous solution of ethanolthe final mixture contains 57.4% Stevioside and 31.9% Rebaudioside A.Further re-crystallization from 90% aqueous solution of ethanol resultedin product with higher content of Rebaudioside A. The purity of theproduct was around 80%. Stevioside further can be purified up to 86.1%by additional washing with water. β-Type crystals of Stevioside andα-type crystals of Rebaudioside A were obtained.

JP No. 57-046998 concerns the preparation of Stevioside. Raw leaves ofStevia rebaudiana were extracted with 10-20 volumes of water and thefiltrate was treated with calcium hydroxide in an amount of 10-30% ofthe raw leaves weight. The pH of the suspension was then adjusted to 4-6with sulfuric acid or citric acid. After filtration the extract waspassed through a polyimide column to adsorb glycosides and removeimpurities. The purified extract was then concentrated under reducedpressure, pH adjusted to 8-9 with aqueous ammonia and extracted withn-butanol to afford crude Stevioside, which was then recrystallized frommethanol.

JP No. 57-086264 concerns the isolation of principal sweeteningcomponent of Stevia. Dried stalks and leaves of Stevia were extractedwith cold water, hot water, hydrous alcohol, etc. The extract wascoagulated or precipitated with an adsorbent, and the precipitate wasremoved by filtration or centrifugation to obtain a clear liquidcontaining the sweetening components. The components were adsorbed on asynthetic polymer adsorbent, purified to 80-90% purity, concentrated,dried, and dissolved in 3-8 volumes of hot methanol or hot ethanol.Stevioside and Rebaudioside A were crystallized from the solutionsimultaneously. After complete removal of the solvent, the mixedcrystals were heated together with a 3-6 volumes of alcohol and thesolids were separated from solution by hot filtration. Stevioside can beobtained from the solution and the Rebaudioside A can be prepared bywashing and drying the solid part.

JP No. 06-192283 and JP No. 08-000214 discloses purification ofRebaudioside A by gel-filtration on Toyo Perl HW-40. Rebaudioside C andDulcoside were obtained by HPLC. Method is useful only in laboratoryscale.

JP 63173531 describes a method of extracting sweet glycosides from theStevia rebaudiana plant. The first step of the process was to extract aliquid solution of sweet glycosides from the Stevia rebaudiana plant.Secondly, the liquid solution of sweet glycosides was passed through anon-polar porous resin and eluted with a water-soluble organic solvent,preferably methanol. Thirdly, the eluted solution was concentrated anddried to give a powdery material. This procedure isolates a mixture ofsweet glycosides, but does not isolate a single pure sweet glycosidesuch as Rebaudioside A.

JP No. 07-143860 discloses purification of Rebaudioside A throughcrystallization and re-crystallization from 10-20% of aqueous methanolsolution. The purity of Rebaudioside A was around 90%.

JP No. 07-177862 discloses purification of Rebaudioside A andStevioside. Purified Stevia extract was treated with low concentrationsof alcohol to obtain crystals with about 75% content of Stevioside andRebaudioside A. The crystals were further recrystallized from water toprovide the slightly water-soluble sweetener with ratio of Steviosideand Rebaudioside A around 1:2, w/w.

JP No. 2002,262,822 discloses a sweetener extracted from dried leaves ofStevia plant and its extraction method. This process used water oraqueous solvent to extract Stevia glycosides from the dried leaves. Inthe obtained product, the content of Rebaudioside A is 2.56 times theamount of Stevioside.

Isolation of Steviolbioside, Rebaudioside A, Rebaidioside B was carriedout by Kohda et al., 1976. Dried leaves were extracted with hot methanoland filtrate was concentrated to dryness. The residue was extracted withn-butanol and, after drying the residue was re-crystallized frommethanol. The mother liquor was subjected to chromatographic separationon silica gel using chloroform-methanol-water mixture as mobile phase.Further purification was developed by thin-layer chromatography. Themethod can be applied only in laboratory scale for the production ofsmall amounts of abovementioned sweet glycosides.

Dulcoside A and B were isolated and identified using crystallizationfrom methanol-ethanol mixture and further purified by chromatography onsilica gel (Kobayashi et al., 1977).

The combination of ultrafiltration, diafiltration, reverse osmosis andion exchange treatment was used for the purification of Stevia extract(Fuh and Chiang, 1990). The cutting capacity of ultrafiltrationmembranes was 25,000 and 100,000 Daltons. The mixtures of strong andweak cation- and anion-exchange resins were used as ion-exchangers. Therecovery of total steviol glycosides was around 90%; however the finalproduct purity was 46% only.

A method for the purification of steviol glycosides by membranetechnology is described by Liu et al. (1991) and Zhang et al. (2000).Dried leaves were placed in a standard glass column and extraction wascarried out with reverse osmosis water. The extract was pretreated witha ceramic tubular membrane and then with an ultrafiltration membrane indiafiltration mode. Permeate was washed from lower molecular weightimpurities by a nanofiltration membrane in a diafiltration mode atelevated temperatures. Addition of lime and/or other flocculating agentto ultrafiltration feed improved the flux significantly. The processcould provide a relatively high purity sweetener concentrate. Howeverthere are no data about the purity of the extract and the recovery ofsteviol glycosides. The low pH values used for the extraction requiredspecial acid resistant reactors. Low temperatures during extractionincreased the operational cost of the production. These both (lowtemperatures and pH) resulted in large amount of diluted initialextract. Dilution of extract occurred also during microfiltration andultrafiltration. For the final purification ion-exchange treatment isnecessary. These factors are substantially increasing the productioncost and decreasing the yield of final product in unit of time. Initialinvestment is high as well.

A series of polar resins based on polystyrene with carbonyl groups wereused for the adsorption of steviol glycosides and partial separation ofStevioside and Rebaudioside A (Chen et al., 1998; 1999). The ratio ofRebaudioside A to Stevioside can increase from 0.72 to 2.24.

The adsorptive capacity and selectivity of a novel adsorbent withpyridyl group toward steviol glycosides were studied (Chen et al.,1999). The effect of polarity and physical structure of the sorbent onthe selectivity was investigated in detail. Two separation methods wereapplied in the enrichment of Rebaudioside A. They were selective elutionusing methanol or ethanol solution as solvent, and dynamicchromatographic separation using pyridyl resin with high selectivity.Results show that the chromatographic separation method can effectivelyenrich Rebaudioside A from Stevia extract with high content ofStevioside. The ratio of Rebaudioside to Stevioside can increase from0.75 to 3.94. Further purification of Rebaudioside A was possible bycrystallization from methanol.

A method for clarification of Stevia extract using modified zeolites isdescribed by Moraes et al. (2001) and Montovaneli et al. (2004).Synthetic or natural zeolites were modified by treatment with calcium orbarium ions and Stevia extract without any pretreatment was contactedwith modified zeolite. It resulted in 70-80% of clarification in batchand only 55-60% in continuous conditions. The clarification process wasmeant to adsorb the pigments that make the extract brownish, and not theglycosides, which are responsible for the sweet taste. However, thereare no data on the steviol glycosides content in the final product.Obviously only this type of treatment cannot result in highly purifiedextract, especially because of polysaccharides, heavy metals andsterebins, which remain in clarified extract. Moreover, no data abouthalf-life and adsorption capacity of the carrier which is very importantwhen process is carried out in continuous conditions.

Polymeric adsorbents with (CH₃)₃ groups were designed and applied forthe purification of steviol glycosides and enrichment of Rebaudioside A(Shi et al., 2002). In the series of five columns the content ofRebaudioside A increased from the first column product to the fifthcolumn product. At the same time the adsorbent displayed decolorizationability.

Rebaudioside F was isolated by liquid chromatography on3-aminopropyl-functionalized silica gel by Starratt et al., 2002. Thefractions which were rich in Rebaudioside C and Rebaudioside F werecombined and separated by HPLC on a Waters carbohydrate column withlinear gradient of acetonitrile and water.

Preparation of Stevia extract by supercritical fluid extraction isdescribed by Yoda et al. 2003. It is a two-step process: (i) CO₂extraction at 200 bar and 30° C., and (ii) CO₂+water extraction.Approximately 72% of the CO₂-soluble compounds were recovered and themajor compound was austroinulin. The system Stevia+CO₂+water was able toremove approximately 50% of the original Stevioside and about 72% ofRebaudioside A. The main drawbacks of the method are the requirement ofhigh pressure and low extraction rate of sweet compounds. Besides, thereis no information about content of minor compounds and total steviolglycosides content in the final extract. The process is difficult toapply on commercial scale.

Pressurized fluid extraction using water or methanol was employed forthe extraction of Stevioside from Stevia rebaudiana Bertoni (Pol et al.(2007). A temperature of 110° C. was determined to be optimal forextraction of Stevioside from Stevia rebaudiana leaves using eitherwater or methanol. An increased temperature resulted in significantdegradation of Stevioside in the media of both solvents or in a declinein the extraction yield in water. Both solvents demonstrated Steviosideextraction with very similar reproducibility and the proposed extractionparameters are the same for both methods. Water represents a moreenvironmentally conscious and cheaper alternative to methanol.

A method of preparation and purification of Stevia extract is describedby Kovylyaeva et al., 2007. The method included extraction of dry leaveswith 14 volumes of distilled water for 1 hrs at boiling temperatures,filtration and concentration of filtrate up to syrup state. Syrup wasdiluted, added AlCl₃.6H₂O and stirred until it dissolved. The mixturewas stirred and treated with a water solution of NaOH. The precipitatewas filtered and the filtrate was passed through a column packed withAl₂O₃. The column was eluted with distilled water to obtain a lightbrown solution. Further purification of Stevioside, Rebaudioside A, andRebaudioside C was done by extraction with n-butanol and columnchromatography on Al₂O₃ and silica gel. The method is unable to resultin high purity Stevia extract. Large amount of salts are used for thepretreatment. Purification process is difficult to apply on commercialscale.

An efficient microwave-assisted extraction process of Stevioside andRebaudioside A is described by Bandna et al. (2009). Dried and powderedleaves of S. rehaudiana were extracted by conventional, ultrasound andmicrowave-assisted extraction techniques using methanol, ethanol andwater as single solvents as well as in binary mixtures. Conventionalcold extraction was performed at 25° C. for 12 h while ultrasoundextraction was carried out at temperature of 35±5° C. for 30 min.Microwave-assisted extraction was carried out at a power level of 80 Wfor 1 min at 50° C. As a result microwave-assisted extraction yielded8.64% and 2.34% of Stevioside and Rebaudioside A, respectively, whileconventional and ultrasound techniques yielded 6.54 and 1.20%, and 4.20and 1.98% of Stevioside and Rebaudioside-A, respectively.

The efficient isolation of steviol glycosides was achieved also usingpressurized hot water extraction (Teo et al., 2009).

All the existing methods deal with isolation and purification of one orother steviol glycoside from the initial extract and do not show a wayfor the further treatment of residual solution or purification of minorcompounds. Thus, there is a need for efficient and economical method forcomprehensive retreatment of extract produced from Stevia rebaudianaBertoni plant.

However, there is no published data on the commercial isolation andpurification of Rebaudioside D which possess excellent sensoryproperties.

Rebaudioside D content in the extract is very low and because of thatits purification is very difficult.

Accordingly, there is a need for a simple, efficient, and economicalmethod for production of high purity Rebaudioside D, which can be usedas sweetener in food, beverage, pharmaceutical, cosmetic, and otherindustries.

SUMMARY OF THE INVENTION

The invention relates to a process for isolation and purification ofindividual sweet glycosides from Stevia rebaudiana Bertoni plant, andmore particularly for isolation and purification of Rebaudioside D andits application in low-calorie fruit juice.

The primary technical problem to be solved and the primary object of theinvention are to provide a highly efficient method of isolating andpurifying different steviol glycosides particularly Rebaudioside D fromStevia extract.

The present invention provides a process for retreatment of Steviarebaudiana Bertoni plant extract with isolation and purification ofhighly purified individual sweet glycosides, particularly RebaudiosideD.

The highly purified Rebaudioside D alone or in the combination withother sweeteners and/or other ingredients is useful as non-caloricsweetener in edible and chewable compositions such as any beverages,confectionaries, bakeries, cookies, chewing gums, and alike.

According to the present invention the isolation and purification ofRebaudioside D was developed from Stevia extract. In one embodiment, themethod for isolation and purification of Rebaudioside D comprisestreating Stevia extract with a first alcohol or alcohol-water solutionto form a first mixture, obtaining a first precipitate containingRebaudioside A and Rebaudioside D from the first mixture, treating thefirst precipitate with a second alcohol or alcohol-water solution toform a second mixture, obtaining from the second mixture a secondprecipitate containing highly purified Rebaudioside A and a filtratewith high Rebaudioside D content, where the second precipitate is driedto produce high purity Rebaudioside A, and where the filtrate isconcentrated to produce high purity Rebaudioside D. Optionally, themethod further comprises treating the purified Rebaudioside D with athird alcohol or alcohol-water solution to refine the high purityRebaudioside D.

An object of the invention is to provide a fermented food composition,particularly, low calorie soya sauce, having excellent taste profile,mouthfeel and physical characteristics. The product comprises at leastone non-nutritive natural sweetener in an amount sufficient to provideperceptible sweetening. The composition provides a more sugar-like tasteprofile due to use of rebaudioside D as a non-nutritive naturalsweetener in an amount sufficient to provide perceptible sweetening.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory and areintended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of the invention. The drawings illustrate embodiments ofthe invention and together with the description serve to explain theprinciples of the embodiments of the invention.

FIG. 1 shows the chemical structure of steviol and the steviolglycosides present in the Stevia rebaudiana Bertoni leaves.

FIG. 2 shows the chemical structures of steviol glycosides present inStevia rebaudiana Bertoni.

FIG. 3 shows one-stage purification scheme of Rebaudioside A usingethanol-water systems in accordance with one embodiment of the presentinvention.

FIG. 4 shows the HPLC charts of Rebaudioside D at various stages ofpurification.

FIG. 5 shows a purification scheme of Rebaudioside D in accordance withone embodiment of the present invention.

FIG. 6 shows FTIR spectrum of Rebaudioside D.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a process for isolation and purificationof individual sweet glycosides from Stevia rebaudiana Bertoni plantextract, and more particularly for isolation and purification ofRebaudioside D from Stevia rebaudiana Bertoni plant extract and itsapplication in low-calorie fruit juice.

Advantages of the present invention will become more apparent from thedetailed description given hereinafter. However, it should be understoodthat the detailed description and specific examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly, since various changes and modifications within the spirit andscope of the invention will become apparent to those skilled in the artfrom this detailed description.

Among sweet glycosides existing in Stevia only Stevioside andRebaudioside A are available at moderate cost at <80% purity and at highcost at >80% purity. The highest purity of commercial product usually ismore than 97%. In the market there are no commercial quantities forRebaudioside B, Rebaudioside D, and Rebaudioside C. Rebaudiosides E andF analytical standards in minor quantities are still unavailable.

Rebaudioside D is a high-potency diterpenoid glycoside sweetener havingthe chemical structure presented in FIG. 2.

Rebaudioside D is isolated and extracted, along with other steviolglycosides, from the Stevia rebaudiana Bertoni plant (“Stevia”), whichis commercially cultivated in Japan, Taiwan, Malaysia, South Korea,China, Israel, India, Brazil, Australia, and Paraguay. It is an idealnon-caloric sweetener with functional and sensory properties superior tothose of many high-potency sweeteners. Processed forms of Stevia can be30 to 400 times more potent than sugar. Amongst the sweet diterpenoidglycosides of Stevia, Rebaudioside D is the least bitter, and with theleast persistent aftertaste.

At present there is no published commercial technology related to theisolation and purification of Rebaudioside D, and certainly there is aneed for efficient and economical method for comprehensive isolation andpurification of individual sweet glycosides from Stevia extract.

The present invention provides a method for production of highlypurified Rebaudioside D from Stevia extract.

Hereinafter, the term “highly purified” refers to a Rebaudioside Dcomposition that includes at least about 91% to 100% of the RebaudiosideD on dry weight basis.

Exemplary embodiments of this invention are described in detail belowand illustrated in FIGS. 3-6.

However, in the detailed description, only certain exemplary embodimentsof the present invention are shown and described, by way ofillustration. As those skilled in art will recognize, the invention canbe embodied in many different forms and should not be construed as beinglimited to the embodiments set forth herein.

Diterpene glycosides, including sweet-tasting substances, are found inthe stems, seeds and leaves of the S. rebaudioside Bertoni plant, beingpresent in the highest concentration in the leaves. The leaves,therefore, are the preferred starting material for recovery of sweetglycosides.

Rebaudioside D purification is developed starting from commercial Steviaextract. The content of Rebaudioside D in the extract can vary dependingon Stevia plant variety or technological scheme of the extractpreparation.

Stevia extract containing Stevioside −25.40%, Rebaudioside A −59.14%,Rebaudioside C −9.71%, Rebaudioside D −2.03%, Rebaudioside B −0.56%,Rebaudioside E −0.68%, Rebaudioside F −1.02%, Steviolbioside −0.11%, andDulcoside A −1.35% was used as an exemplary starting material inillustrating the purification of Rebaudioside D.

Now referring to FIG. 3, there is provided a one-stage purification ofhigh purity Rebaudioside A with relatively high Rebaudioside D inaccordance with one embodiment of the present invention. Stevia extractwas dissolved in a first ethanol-water solution at 50-70° C., preferably55-60° C., for about 10-30 min, preferably 15-20 min, and then at 15-40°C., preferably 20-22° C. for about 18-48 hours, preferably 20-24 hourswith agitation. When the temperature reached to 22° C., 1-2% (w/vol.) ofhighly purified Rebaudioside A was added to the reaction mixture as astarter to initiate crystallization. The proportion of extract and thefirst ethanol-water solution depended on the content of minor glycosidesand was between 1.0:2.5-1.0:10.0, w/v, preferably 1.0:3.0-5.0, w/v.

During this time a first precipitate was formed, which was separated byfiltration or centrifugation.

The concentration of ethanol in the first ethanol-water solution isbetween 75-99%, preferably 82-88%. The content of Rebaudioside A andRebaudioside D in the first precipitate ranges between 79-99% and0.8-4.0% respectively.

The purity and yield of Rebaudioside A depended on the ration of extractto ethanol-water solution and concentration of ethanol. The data atvarious concentrations of ethanol is summarized in the TABLE 3. Theextract:methanol ratio was 1:3.0, w/v.

The purification level and output of Rebaudioside A at various volumesof 88% ethanol solution is summarized in the TABLE 4.

TABLE 3 Steviol glycosides, % Ethanol, % St RebA RebC RebD RebB RebERebF StBio DulA Yield, % 75 0.1 98.9 0.2 0.8 0 0 0 0 0 19.2 78 0.1 98.60.2 1.0 0.1 0 0 0 0 21.3 80 0.1 98.2 0.2 1.4 0.1 0 0 0 0 23.4 82 0.197.8 0.2 1.8 0.1 0 0 0 0 23.7 85 0.1 97.6 0.2 2 0.1 0 0 0 0 24.1 87 0.396.7 0.4 2.5 0.1 0 0 0 0 25.6 88 0.4 95.6 0.3 3.5 0.1 0.1 0 0 0 33.0 890.8 94.2 0.7 3.5 0.2 0.1 0.2 0 0.3 35.4 90 1.4 93.4 1.2 3.0 0.2 0.1 0.20.1 0.4 35.7 95 3.2 90.0 3.1 2.5 0.2 0.2 0.2 0.1 0.5 41.6 99 7.2 78.810.3 2.1 0.2 0.3 0.2 0.2 0.7 48.3

TABLE 4 Extract/Ethanol Steviol glycosides, % ratio, w/v St RebA RebCRebD RebB RebE RebF StBio DulA Yield, % 1:5.0 0.2 98.0 0.2 1.5 0 0.1 0 00 26.5 1:4.0 0.2 97.5 0.2 2.0 0 0.1 0 0 0 31.3 1:3.5 0.3 96.9 0.1 2.6 00.1 0 0 0 32.4 1:3.0 0.4 95.6 0.3 3.5 0.1 0.1 0 0 0 33.0 1:2.5 2.5 91.71.7 3.3 0.2 0.2 0.1 0 0.3 35.6 1:2.0 3.3 89.8 2.5 3.2 0.2 0.3 0.1 0.10.5 41.4

The Rebaudioside D content increases with the increase of theconcentration of ethanol up to 88-90% and the decrease of the ration ofethanol-water solution to extract. At the same time the purity ofRebaudioside A increased with more diluted ethanol solutions and higherratios of ethanol-water solution to extract.

The yield of the product at this stage for Stevia extracts with variouscontents of Rebaudioside A, after treatment with 1:3 (w/vol.) ratios of88% ethanol is summarized in the TABLE 5. As it could be expected theyield of the product increases with increase of the content ofRebaudioside A in the initial extract.

TABLE 5 Rebaudioside A content in initial Yield of Rebaudioside A atprecipitation extract, % stage from initial extract, % 42.0-43.022.0-25.0 45.0-46.0 22.0-25.0 50.0-53.0 24.0-27.0 55.0-59.0 28.0-31.060.0-62.0 32.0-36.0

The precipitate was separated by filtration or centrifugation, washedwith about two volumes of absolute ethanol and dried. Any type ofequipment which allows separation of precipitate from liquid, such asvarious types of centrifuges or filtration systems can be used in thisstage. Different type dryers, such as rotary vacuum dryer, fluid beddryer, rotary tunnel dryer or plate dryer, are suitable to producepurified steviol glycosides in powder form.

In case if initial extract contains high amount of Rebaudioside B andRebaudioside D, for Rebaudioside A and later Rebaudioside D purificationlower concentrations of ethanol and higher ratio of ethanol-watersolution to the extract are preferred to use (TABLE 6; TABLE 7). In thisseries of experiments the Rebaudioside A content in the initial extractwas 48.7%.

The yield of the product with high content of Rebaudioside A andRebaudioside I) can be increased by using ethanol forafter-precipitation. For that purpose at the end of crystallization,0.5-1.0, v/w, preferably 0.5-0.8, v/w, of absolute ethanol to theinitial solid, was added to the mixture and the process was continuedfor another 2-3 hours. The yield and purity of the product from extractwith 48.7% of Rebaudioside A content are summarized in TABLE 8.

TABLE 6 Purity of product at different Rebaudioside B content, %(Rebaudioside D Ratio ethanol content was 0.4%) Ethanol, % to solid, v/w0% 0.4% 0.8% 1.1% 81.0 2.5 98.7 98.5 98.2 97.9 3.0 98.9 98.7 98.4 98.13.5 99.2 98.9 98.6 98.4 83.0 2.5 98.1 98.2 98.0 97.7 3.0 98.5 98.4 98.297.9 3.5 98.8 98.6 98.4 98.2 85.0 2.5 97.7 97.6 97.4 97.2 3.0 98.2 97.997.6 97.4 3.5 98.5 98.2 97.8 97.6 87.0 2.5 96.3 97.2 96.6 96.4 3.0 97.597.6 97.4 97.0 3.5 97.9 97.9 97.6 97.2 88.0 2.5 96.1 95.9 95.5 95.1 3.097.3 97.1 96.4 95.8 3.5 97.7 97.5 97.2 96.8 90.0 2.5 94.6 94.1 92.3 90.53.0 96.3 95.8 92.8 91.2 3.5 97.3 96.8 93.7 91.9

TABLE 7 Purity of product at different content of Rebaudioside D, %(Rebaudioside B Ratio ethanol content was 0.1%) Ethanol, % to solid, v/w0.5% 1.2% 1.7% 2.6% 81.0 2.5 98.7 98.0 97.5 97.1 3.0 98.9 98.3 98.0 97.43.5 99.2 98.5 98.2 97.7 83.0 2.5 98.1 97.7 97.3 97.0 3.0 98.5 98.1 97.897.4 3.5 98.8 98.4 98.0 97.6 85.0 2.5 97.7 97.5 97.1 96.8 3.0 98.2 97.897.6 97.1 3.5 98.5 98.1 97.8 97.2 87.0 2.5 96.3 96.2 95.8 94.2 3.0 97.597.3 96.7 96.1 3.5 97.9 97.6 97.4 96.9 88.0 2.5 96.1 95.7 95.2 93.7 3.097.3 97.1 96.5 95.6 3.5 97.7 97.4 97.0 96.6 90.0 2.5 94.6 94.2 93.5 93.03.0 94.8 94.8 93.9 93.3 3.5 95.7 95.4 94.4 93.5

TABLE 8 Yield and purity of RebA at different concentrations of ethanol(ratio of ethanol to extract = 1:3.5, w/v) Additional 85% 86% 87% 88%ethanol volume, Yield, RebA, Yield, RebA, Yield, RebA, Yield, RebA, v/wto solids % % % % % % % % 0 29.5 98.5 30.6 98.3 32.7 97.9 33.3 97.8 0.531.4 98.5 31.6 98.2 33.4 97.9 33.8 97.6 0.6 32.3 98.2 32.7 98.2 34.397.8 34.7 97.6 0.7 33.5 97.9 33.9 97.7 35.4 97.6 35.9 97.5 0.8 34.1 97.935.2 97.7 36.3 97.6 36.7 97.4 0.9 34.3 97.8 35.4 97.6 36.7 97.5 37.497.4 1.0 34.5 97.8 35.7 97.5 36.9 97.4 37.7 97.2

To produce high purity Rebaudioside A the process can be carried out at30-50° C. without cooling stage. Although the purity of Rebaudioside Awas higher it resulted in lower yield of the product. The quality of theproduct increased at higher washing temperatures. The results obtainedusing 3.5 volumes of 85% ethanol to one part of extract after 24 hoursand with and without after-precipitation stage are summarized in TABLE9.

TABLE 9 Yield, % Content of Rebaudioside A With after- With after-Without precipitation Without precipitation Temperature, after- (0.8vol. after- (0.8 vol. ° C. precipitation EtOH) precipitation EtOH) 22.029.6 33.5 98.2 98.5 30.0 28.7 32.8 98.4 98.6 35.0 27.5 32.2 98.7 98.940.0 27.0 31.4 98.8 99.2 45.0 25.4 28.9 99.0 99.4 50.0 24.3 25.6 99.299.5 Rebaudioside A, Rebaudioside B and Rebaudioside D contents were51.3, 0.2% and 0.7%, respectively.

When the content of Rebaudioside A in the final product was less than97% mainly due to high content of Rebaudioside B and/or Rebaudioside D,the product was additionally washed with aqueous solution of ethanol.For that the Rebaudioside A obtained after the precipitation wassuspended in the ethanol-water mixture at room temperature for 30-40min. After homogeneous suspension was obtained the temperature wasincreased up to 35-50° C. preferably 38-42° C. and agitated for about10-20 hours, preferably 12-15 hours, and then at 10-25° C., preferably20-22° C. for about 3-20 hours, preferably 5-10 hours. The proportion ofRebaudioside A and ethanol was 1.0:2.0-1.0:5.0, w/v, preferably1.0:2.5-4.0, w/v. The ethanol concentration was between 85-93%preferably 88-90%.

In case if purity of Rebaudioside A was lower than 97% due to highcontent of Stevioside, the product was washed with absolute ethanol bythe same way as it was described above for Rebaudioside B andRebaudioside D contaminated product. The proportion of Rebaudioside Aand ethanol was 1.0:2.0-1.0:5.0, w/v, preferably 1.0:2.5-4.0, w/v.

Now referring to FIG. 5, there is provided a functional flowchart forpurification of Rebaudioside A and Rebaudioside D in accordance with oneembodiment of the present invention.

Purification of Rebaudioside D from the crystals/precipitates withRebaudioside A and Rebaudioside D content around 75-80% and 2.0-3.5%respectively, was developed as follows. It is to be noted that thecrystals with Rebaudioside A and Rebaudioside D can be obtained from theprocess as described in connection with FIG. 3.

The precipitate with high content of Rebaudioside A and Rebaudioside Dwas mixed with a second ethanol-water solution and incubated at 45-65°C. preferably 50-55° C. for 2-6 hours preferably 3-4 hours withagitation. Then, the mixture was cooled down to room temperatures for1-3 hour preferably 0.5-1.0 hour. The precipitate was separated byfiltration.

The preferable ratio of solids to aqueous ethanol solution was 1 to 5,w/v, and the optimum concentration of ethanol was 78%. However ethanolconcentration can be in the limits 70-80% and ratio 1:2.5-1.7, w/v.

To facilitate the filtration of high Rebaudioside D precipitate,activated carbon in amount of 0.5-3.0 vol. % preferably 1.0-1.5 vol. %was added to the mixture before filtration. The precipitate was thenmixed with 3-5 volumes of 30-50% of methanol. The suspension wasmaintained with agitation at 45-65° C. preferably 57-62° C. for 1-5hours preferably 2-3 hours and subjected to filtration. Elution ofadsorbed on activated carbon glycosides was carried out with methanol.

Both precipitates obtained without and with carbon application contain19-22.1% of Rebaudioside D at the optimal conditions (TABLE 10).

TABLE 10 Ratio ethanol to Purity of Purity of Ethanol, % solid, v/wRebA, % RebD, % 75.0 4.0 98.5 18.4 5.0 99.2 18.6 6.0 99.4 18.6 77.0 4.098.4 18.7 5.0 99.1 20.1 6.0 99.2 20.3 78.0 4.0 98.4 19.2 5.0 99.2 22.06.0 99.4 22.1 79.0 4.0 98.1 19.0 5.0 98.8 19.7 6.0 99.0 19.8 80.0 4.098.0 17.3 5.0 98.4 17.9 6.0 98.9 18.2 82.0 4.0 97.7 15.2 5.0 98.1 15.86.0 98.7 16.4

In principle the higher the applied volume of methanol the faster can beelution process. The process can be completed in shorter time periodwhen aqueous solution of methanol was used.

The methanol fraction was evaporated to dryness.

When the initial material containing 95.6% of Rebaudioside A and 3.5%Rebaudioside D (FIG. 4 a) was mixed with 3.5 volumes of 78.0% ofethanol, the mixture was boiled for 10-15 min and undissolved materialwas separated by hot filtration, the output of precipitate was in thelimits of 6-7.0% with 52-53.0% and 43-45.0% of Rebaudioside A andRebaudioside D (FIG. 4 b) contents, respectively.

For the further purification the precipitate was suspended in 50%ethanol at the ratio of 1:2, w/v and at 30-40° C. preferably 33-37° C.,and maintained for 2-15 hours preferably 10-12 hours with agitation. Thesuspension was filtered and dried. The yield of precipitate with contentof about 15-17.0% Rebaudioside A and 80-82% Rebaudioside D was in therange of 42-46.0%. In principle up to five volumes of aqueous ethanolcan be applied at this stage. The concentration of ethanol can be in thelimits of 10-80% preferably 45-52%.

The precipitate was subjected to similar treatment. The precipitate wasseparated by filtration, washed with about two volumes of anhydrousmethanol and dried. Any type of equipment, which allows separatingprecipitate from liquid, such as various type centrifuges or filtrationsystems, can be used in this stage. Different type of dryers, such asrotary vacuum dryer, fluid bed dryer, rotary tunnel dryer or plate dryerare suitable to produce purified Rebaudioside D in powder form.

The purity of Rebaudioside D was around 95-99% content (FIG. 4 c). Theyield of the product was around 58-60%.

The remaining combined solution after isolation of Rebaudioside D wasmixed with small amount of Rebaudioside A as starter and left forcrystallization at 20-22° C. for 20-24 hours. Rebaudioside A content inthe crystals ranged 97.7-99.4%.

The remaining solution from the first precipitation can be used forisolation of Rebaudioside A or highly purified mixture of steviolglycosides.

High purity Rebaudioside D obtained in this invention has 1129.15molecular weight, C₅₀H₈₀O₂₈ molecular formula and structure presented inthe FIG. 2, and is in the form of white and odorless powder. Thecompound is about 180-200 times sweeter than sugar when compared to 10%sucrose solution. The infrared absorption spectrum is shown in the FIG.6. Rebaudioside D exhibits a characteristic absorption maximum at around1730 cm⁻¹. Other properties of the pure Rebaudioside D are as follows:

Melting point: 248-249° C.

Specific rotation: [α]_(D) ²⁵ −29.5° in 50% ethanol (C=1.0).

Solubility in water is around 0.2% which is increasing with increase intemperature. It precipitates again upon cooling the solution. Highlysoluble during chromatographic separation stage and beforecrystallizing.

Soluble in diluted solutions of methanol, ethanol, n-propanol, andisopropanol.

Insoluble in acetone, benzene, chloroform, and ether.

Rebaudioside D obtained in this invention is heat and pH-stable.

Rebaudioside D obtained according to this invention may be incorporatedas a high intensity natural sweetener in foodstuffs, beverages,pharmaceutical compositions, cosmetics, chewing gums, table topproducts, cereals, dairy products, toothpastes and other oral cavitycompositions, etc. The examples which follow show representativeproportions which may be employed.

In addition, Rebaudioside D can be used as a sweetener not only fordrinks, foodstuffs, and other products dedicated for human consumption,but also in animal feed and fodder with improved characteristics.

During the manufacturing of foodstuffs, drinks, pharmaceuticals,cosmetics, table top products, chewing gum the conventional methods suchas mixing, kneading, dissolution, pickling, permeation, percolation,sprinkling, atomizing, infusing and other methods can be used.

The sweetener obtained in this invention can be used in dry or liquidforms. It can be added before or after heat treatment of food products.The amount of the sweetener depends on the purpose of usage. It can beadded alone or in the combination with other compounds.

In one particular embodiment of this invention Rebaudioside D, as asweetening compound, may be employed as the sole sweetener, or it may beused together with other naturally occurring high intensity sweeteners.

The phrase “natural high intensity sweeteners”, as used herein, refersto any compositions which are found in nature and which have sweetnesspotency higher than sucrose, fructose, or glucose.

Non-limiting examples of natural high intensity sweeteners includeStevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, RebaudiosideE, Rebaudioside F, Steviolbioside, Dulcoside A, Rubusoside, mogrosides,brazzein, glycyrrhizic acid and its salts, thaumatin, perillartine,pernandulcin, mukuroziosides, baiyunoside, phlomisoside-I,dimethyl-hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin,carnosiflosides, cyclocarioside, pterocaryosides, polypodoside A,brazilin, hernandulcin, phillodulcin, glycyphyllin, phlorizin,trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastilibin,trans-cinnamaldehyde, monatin and its salts, selligueain A, hematoxylin,monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin,pentadin, miraculin, curculin, neoculin, chlorogenic acid, cynarin, LuoHan Guo sweetener, siamenoside and alike, and combinations thereof.

In another particular embodiment Rebaudioside D as a sweetening compoundmay be used together with synthetic or artificial high intensitysweeteners.

The phrase “synthetic” or “artificial high intensity sweeteners”, asused herein, refers to any compositions which are not found in natureand which have—sweetness potency higher than sucrose, fructose, orglucose.

Non-limiting examples of synthetic or artificial high intensitysweeteners include sucralose, potassium acesulfame, aspartame, alitame,saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, dulcin,suosan,N—[N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine1-methyl ester,N—[N-[3-(3-hydroxy-4-methoxyphenyl)-3-methylbutyl]-L-α-aspartyl]-L-phenylalanine1-methyl ester,N—[N-[3-(3-methoxy-4-hydroxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine1-methyl ester, salts thereof, and the like, and combinations thereof.

In one embodiment Rebaudioside D can be used in the combination withnatural sweetener suppressors such as gymnemic acid, hodulcin, ziziphin,lactisole, and the like.

In another embodiment Rebaudioside D can be combined with various umamitaste enhancers.

In a particular embodiment Rebaudioside D can be formulated with aminoacids including, but not limited to, aspartic acid, arginine, glycine,glutamic acid, proline, threonine, theanine, cysteine, cystine, alanine,valine, tyrosine, leucine, isoleucine, asparagine, serine, lysine,histidine, ornithine, methionine, camitine, aminobutyric acid (alpha-,beta-, or gamma-isomers), glutamine, hydroxyproline, taurine, norvaline,sarcosine, and their salt forms such as sodium or potassium salts oracid salts. The amino acid additives also may be in the D- orL-configuration and in the mono-, di-, or tri-form of the same ordifferent amino acids. Additionally, the amino acids may be α-, β-, γ-,δ-, and ε-isomers if appropriate. Combinations of the foregoing aminoacids and their corresponding salts (e.g., sodium, potassium, calcium,magnesium salts or other alkali or alkaline earth metal salts thereof,or acid salts) also are suitable additives. The amino acids may benatural or synthetic. The amino acids also may be modified. Modifiedamino acids refers to any amino acid wherein at least one atom has beenadded, removed, substituted, or combinations thereof (e.g., N-alkylamino acid, N-acyl amino acid, or N-methyl amino acid). Non-limitingexamples of modified amino acids include amino acid derivatives such astrimethyl glycine, N-methyl-glycine, and N-methyl-alanine. As usedherein, amino acids encompass both modified and unmodified amino acids.As used herein, modified amino acid also may encompass peptides andpolypeptides (e.g., dipeptides, tripeptides, tetrapeptides, andpentapeptides) such as glutathione and L-alanyl-L-glutamine.

In one particular embodiment Rebaudioside D may be formulated withpolyamino acid additives include poly-L-aspartic acid, poly-L-lysine(e.g., poly-L-α-lysine or poly-L-ε-lysine), poly-L-ornithine (e.g.,poly-L-α-ornithine or poly-L-ε-ornithine), poly-L-arginine, otherpolymeric forms of amino acids, and salt forms thereof (e.g., magnesium,calcium, potassium, or sodium salts such as L-glutamic acid mono sodiumsalt). The polyamino acid additives also may be in the D- orL-configuration. Additionally, the polyamino acids may be α-, β-, γ-,δ-, and ε-isomers if appropriate. Combinations of the foregoingpolyamino acids and their corresponding salts (e.g., sodium, potassium,calcium, magnesium salts or other alkali or alkaline earth metal saltsthereof or acid salts) also are suitable sweet taste improving additivesin embodiments of this invention. The polyamino acids described hereinalso may comprise co-polymers of different amino acids. The polyaminoacids may be natural or synthetic. The polyamino acids also may bemodified, such that at least one atom has been added, removed,substituted, or combinations thereof (e.g., N-alkyl polyamino acid orN-acyl polyamino acid). As used herein, polyamino acids encompass bothmodified and unmodified polyamino acids. In accordance with particularembodiments, modified polyamino acids include, but are not limited topolyamino acids of various molecular weights (MW), such aspoly-L-α-lysine with a MW of 1,500, MW of 6,000, MW of 25,200, MW of63,000, MW of 83,000, or MW of 300,000.

In another particular embodiment Rebaudioside D can be combined withpolyols or sugar alcohols. The term “polyol” refers to a molecule thatcontains more than one hydroxyl group. A polyol may be a diol, trial, ora tetraol which contain 2, 3, and 4 hydroxyl groups, respectively. Apolyol also may contain more than four hydroxyl groups, such as apentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7 hydroxylgroups, respectively. Additionally, a polyol also may be a sugaralcohol, polyhydric alcohol, or polyalcohol which is a reduced form ofcarbohydrate, wherein the carbonyl group (aldehyde or ketone, reducingsugar) has been reduced to a primary or secondary hydroxyl group.

Non-limiting examples of polyols include erythritol, maltitol, mannitol,sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol,glycerol, threitol, galactitol, hydrogenated isomaltulose, reducedisomalto-oligosaccharides, reduced xylo-oligosaccharides, reducedgentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup,hydrogenated starch hydrolyzates, polyglycitols and sugar alcohols orany other carbohydrates capable of being reduced which do not adverselyaffect the taste of the sweetener composition, and combinations thereof.

In one particular embodiment Rebaudioside D can be combined with reducedcalorie sweeteners such as D-tagatose, L-sugars, L-sorbose, L-arabinose,and others and combinations thereof.

In another particular embodiment Rebaudioside D can be combined withvarious carbohydrates. The term “carbohydrate” generally refers toaldehyde or ketone compounds substituted with multiple hydroxyl groups,of the general formula (CH₂O)_(n), wherein “n” is 3-30, as well as theiroligomers and polymers. The carbohydrates of the present invention can,in addition, be substituted or deoxygenated at one or more positions.Carbohydrates, as used herein, encompass unmodified carbohydrates,carbohydrate derivatives, substituted carbohydrates, and modifiedcarbohydrates. As used herein, the phrases “carbohydrate derivatives”,“substituted carbohydrate”, and “modified carbohydrates” are synonymous.Modified carbohydrate means any carbohydrate wherein at least one atomhas been added, removed, substituted, or combinations thereof Thus,carbohydrate derivatives or substituted carbohydrates includesubstituted and unsubstituted monosaccharides, disaccharides,oligosaccharides, and polysaccharides. The carbohydrate derivatives orsubstituted carbohydrates optionally can be deoxygenated at anycorresponding C-position, and/or substituted with one or more moietiessuch as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino,amido, carboxyl derivatives, alkylamino, dialkylamino, acylamino,alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl,sulfonyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl,phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino,hydrazino, carbamyl, phospho, phosphonato, or any other viablefunctional group provided the carbohydrate derivative or substitutedcarbohydrate functions to improve the sweet taste of the sweetenercomposition.

Non-limiting examples of carbohydrates in embodiments of this inventioninclude tagatose, trehalose, galactose, rhamnose, various cyclodextrins,cyclic oligosaccharides, various types of maltodextrins, dextran,sucrose, glucose, ribulose, fructose, threose, arabinose, xylose,lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar,isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose,gulose, idose, talose, erythrulose, xylulose, psicose, turanose,cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronicacid, gluconic acid, glucono-lactone, abequose, galactosamine, beetoligosaccharides, isomalto-oligosaccharides (isomaltose, isomaltotriose,panose and the like), xylo-oligosaccharides (xylotriose, xylobiose andthe like), xylo-terminated oligosaccharides, gentio-oligosaccharides(gentiobiose, gentiobiose, gentiotetraose and the like), sorbose,nigero-oligosaccharides, palatinose oligosaccharides,fructooligosaccharides (kestose, nystose and the like), maltotetraol,maltotriol, malto-oligosaccharides (maltotriose, maltotetraose,maltopentaose, maltohexaose, maltoheptaose and the like), starch,inulin, inulo-oligosaccharides, lactulose, melibiose, raffinose, ribose,isomerized liquid sugars such as high fructose corn syrups, couplingsugars, and soybean oligosaccharides. Additionally, the carbohydrates asused herein may be in either the D- or L-configuration. In theformulations any combinations of the compounds can be used.

In a particular embodiment Rebaudioside D may be formulated with sugaracids which is include, but are not limited to, aldonic, uronic,aldaric, alginic, gluconic, glucuronic, glucaric, galactaric,galacturonic, and their salts (e.g., sodium, potassium, calcium,magnesium salts or other physiologically acceptable salts), andcombinations thereof.

In a particular embodiment Rebaudioside D can be used in the combinationwith various physiologically active substances or functionalingredients. Functional ingredients generally are classified intocategories such as carotenoids, dietary fiber, fatty acids, saponins,antioxidants, nutraceuticals, flavonoids, isothiocyanates, phenols,plant sterols and stanols (phytosterols and phytostanols); polyols;prebiotics, probiotics; phytoestrogens; soy protein; sulfides/thiols;amino acids; proteins; vitamins; and minerals. Functional ingredientsalso may be classified based on their health benefits, such ascardiovascular, cholesterol-reducing, and anti-inflammatory.

The composition with Rebaudioside D may include a flavoring agent whichmay be natural or artificial origin. As used herein, unless otherwiseindicated, the term “flavor” means any food-grade material that may beadded to the present compositions to provide a desired flavor to afoodstuff. The flavors useful in the present invention include, forexample, an essential oil, such as an oil derived from a plant or afruit, peppermint oil, spearmint oil, other mint oils, clove oil,cinnamon oil, oil of wintergreen, bay, thyme, cedar leaf, nutmeg,allspice, sage, mace, and almonds. The flavoring agent may be a plantextract or a fruit essence such as apple, banana, watermelon, pear,peach, grape, strawberry, raspberry, cherry, plum, pineapple, apricot,and mixtures thereof. The flavoring agent may be a citrus flavor, suchas an extract, essence, or oil of lemon, lime, orange, tangerine,grapefruit, citron, or kumquat. Flavors useful in the present inventionalso can include cream, hazelnut, vanilla, chocolate, cinnamon, pecan,lemon, lime, raspberry, peach, mango, vanillin, butter, butterscotch,tea, orange, tangerine, caramel, strawberry, banana, grape, plum,cherry, blueberry, pineapple, elderberry, watermelon, bubblegum,cantaloupe, guava, kiwi, papaya, coconut, mint, spearmint, derivatives,and combinations thereof.

The composition with Rebaudioside D may include an aroma component. Asused herein, unless otherwise indicated, the term “aroma component”means any food-grade volatile substance that may be employed to producea desired scent, for example, when mixed with a foodstuff. Aromas usefulin the present invention include, for example, essential oils (citrusoil), expressed oils (orange oil), distilled oils (rose oil), extracts(fruits), anethole (liquorice, anise seed, ouzo, fennel), anisole (aniseseed), benzaldehyde (marzipan, almond), benzyl alcohol (marzipan,almond), camphor (cinnamomum camphora), cinnamaldehyde (cinnamon),citral (citronella oil, lemon oil), d-limonene (orange) ethyl butanoate(pineapple), eugenol (clove oil), furaneol (strawberry), furfural(caramel), linalool (coriander, rose wood), menthol (peppermint), methylbutanoate (apple, pineapple), methyl salicylate (oil of wintergreen),neral (orange flowers), nerolin (orange flowers), pentyl butanoate(pear, apricot), pentyl pentanoate (apple, pineapple), sotolon (maplesyrup, curry, fennugreek), strawberry ketone (strawberry), substitutedpyrazines, e.g., 2-ethoxy-3-isopropylpyrazine;2-methoxy-3-sec-butylpyrazine; and 2-methoxy-3-methylpyrazine (toastedseeds of fenugreek, cumin, and coriander), thujone (juniper, commonsage, Nootka cypress, and wormwood), thymol (camphor-like),trimethylamine (fish), vanillin (vanilla), and combinations thereof.Preferred aroma components according to the present invention include,essential oils (citrus oil), expressed oils (orange oil), distilled oils(rose oil), extracts (fruits), benzaldehyde, d-limonene, furfural,menthol, methyl butanoate, pentyl butanoate, salts, derivatives, andcombinations thereof.

The compositions with Rebaudioside D can comprise a nucleotide additivefor use in embodiments of this invention. They include, but are notlimited to, inosine monophosphate, guanosine monophosphate, adenosinemonophosphate, cytosine monophosphate, uracil monophosphate, inosinediphosphate, guanosine diphosphate, adenosine diphosphate, cytosinediphosphate, uracil diphosphate, inosine triphosphate, guanosinetriphosphate, adenosine triphosphate, cytosine triphosphate, uraciltriphosphate, and their alkali or alkaline earth metal salts, andcombinations thereof The nucleotides described herein also may comprisenucleotide-related additives such as nucleosides or nucleic acid bases(e.g., guanine, cytosine, adenine, thymine, uracil).

The compositions with Rebaudioside D can comprise an organic acidadditive. Organic acids are compounds which comprises a —COOH moiety.Suitable organic acid additives for use in embodiments of this inventioninclude, but are not limited to, C2-C30 carboxylic acids, substitutedhydroxyl C1-C30 carboxylic acids, benzoic acid, substituted benzoicacids (e.g. 2,4-dihydroxybenzoic acid), substituted cinnamic acids,hydroxyacids, substituted hydroxybenzoic acids, substituted cyclohexylcarboxylic acids, tannic acid, lactic acid, tartaric acid, citric acid,gluconic acid, glucoheptonic acids, adipic acid, hydroxycitric acid,malic acid, fruitaric acid (a blend of malic, fumaric, and tartaricacids), fumaric acid, maleic acid, succinic acid, chlorogenic acid,salicylic acid, creatine, glucosamine hydrochloride, glucono deltalactone, caffeic acid, bile acids, acetic acid, ascorbic acid, alginicacid, crythorbic acid, polyglutamic acid, and their alkali or alkalineearth metal salt derivatives thereof. In addition, the organic acidadditives also may be in either the D- or L-configuration.

The compositions with Rebaudioside D can comprise an organic acid saltadditive. They include, but are not limited to, sodium, calcium,potassium, and magnesium salts of all organic acids, such as salts ofcitric acid, malic acid, tartaric acid, flunaric acid, lactic acid(e.g., sodium lactate), alginic acid (e.g., sodium alginate), ascorbicacid (e.g., sodium ascorbate), benzoic acid (e.g., sodium benzoate orpotassium benzoate), and adipic acid. The examples of the sweet tasteimproving organic acid salt additives described optionally may besubstituted with one or more of the following moiety selected from thegroup consisting of hydrogen, alkyl, alkenyl, alkynyl, halo, haloalkyl,carboxyl, acyl, acyloxy, amino, arnido, carboxyl derivatives,alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano,sulfo, thiol, imine, sulfonyl, sulfenyl, sulfinyl, sulfamyl,carboxalkoxy, carboxamide, phosphonyl, phosphinyl, phosphoryl,phosphino, thioester, thioether, anhydride, oximino, hydrazine,carbamyl, phospho, phosphonato, and any other viable functional group,provided the substituted organic acid salt additive functions to improvethe sweet taste of the sweetener composition.

The compositions with Rebaudioside D can comprise an inorganic acidadditive for use in embodiments of this invention. They include, but arenot limited to, phosphoric acid, phosphorous acid, polyphosphoric acid,hydrochloric acid, sulfuric acid, carbonic acid, sodium dihydrogenphosphate, and their corresponding alkali or alkaline earth metal saltsthereof (e.g., inositol hexaphosphate Mg/Ca).

The compositions with Rebaudioside D can comprise a bitter compoundadditive for use in embodiments of this invention, but are not limitedto, caffeine, quinine, urea, bitter orange oil, naringin, quassia, andsalts thereof.

The compositions with Rebaudioside D can comprise an artificial ornatural sweetness enhancers and combinations thereof.

Rebaudioside D formulation may include a polymer additives for use inembodiments of this invention, but are not limited to, chitosan, pectin,pectic, pectinic, polyuronic, polygalacturonic acid, starch, foodhydrocolloid or crude extracts thereof (e.g., gum acacia senegal(Fibergum™), gum acacia seyal, carageenan), poly-L-lysine (e.g.,poly-L-α-lysine or poly-L-ε-lysine), poly-L-ornithine (e.g.,poly-L-α-ornithine or poly-L-ε-ornithine), polyarginine, polypropyleneglycol, polyethylene glycol, poly(ethylene glycol methyl ether),polyaspartic acid, polyglutamic acid, polyethyleneimine, alginic acid,sodium alginate, propylene glycol alginate, sodium hexametaphosphate(SHMP) and its salts, and sodium polyethyleneglycolalginate and othercationic and anionic polymers.

Rebaudioside D formulation may include a protein or protein hydrolyzatesadditives for use in embodiments of this invention, but are not limitedto, bovine serum albumin, whey protein (including fractions orconcentrates thereof such as 90% instant whey protein isolate, 34% wheyprotein, 50% hydrolyzed whey protein, and 80% whey protein concentrate),soluble rice protein, soy protein, protein isolates, proteinhydrolyzates, reaction products of protein hydrolyzates, glycoproteins,and/or proteoglyeans containing amino acids (e.g., glycine, alanine,senrne, threonine, asparagine, glutamine, arginine, valine, isoleucine,leucine, norvaline, methionine, proline, tyrosine, hydroxyproline, andthe like), collagen (e.g., gelatin), partially hydrolyzed collagen(e.g., hydrolyzed fish collagen), and collagen hydrolyzates (e.g.,porcine collagen hydrolyzates).

Rebaudioside D formulation may include a surfactant additives for use inembodiments of this invention, but are not limited to, polysorbates(e.g., polyoxyethylene sorbitan monooleate (polysorbate 80), polysorbate20, polysorbate 60), sodium dodecylbenzenesulfonate, dioctylsulfosuccinate or dioctyl sulfosuccinate sodium, sodium dodecyl sulfate,cetylpyridinium chloride (hexadecylpyridinium chloride),hexadecyltrimethylammonium bromide, sodium cholate, carbamoyl, cholinechloride, sodium glycocholate, sodium taurodeoxycholate, lauricarginate, sodium stearoyl lactylate, sodium taurocholate, lecithins,sucrose oleate esters, sucrose stearate esters, sucrose palmitateesters, sucrose laurate esters, and other emulsifiers, and the like.

Rebaudioside D formulation may include a flavonoid additives for use inembodiments of this invention generally are classified as flavonols,flavones, flavanones, flavan-3-ols, isoflavones, or anthocyanidins.Non-limiting examples of flavonoid additives include catechins (e.g.,green tea extracts), polyphenols, rutins, neohesperidin, naringin,neohesperidin dihydrochalcone, and the like.

The formulation may include an alcohol additives for use in embodimentsof this invention include, but are not limited to, ethanol.

The formulation may include an astringent compound additives include,but are not limited to, tannic acid, europium chloride (EuC₃),gadolinium chloride (GdCl₃), terbium chloride (TbCl₃), alum, tannicacid, and polyphenols (e.g., tea polyphenols).

The formulation may include a vitamin. Vitamins are organic compoundsthat the human body needs in small quantities for normal functioning.The body uses vitamins without breaking them down, unlike othernutrients such as carbohydrates and proteins. The vitamins for use inembodiment include, but not limited to, vitamin A (retinol,retinaldehyde, retinoic acid, retinoids, retinal, retinoic acid),vitamin D (vitamins D1-D5; cholecalciferol, lumisterol, ergocalciferol,dihydrotachysterol, 7-dehydrocholesterol), vitamin E (eocopherol,tocotrienol), vitamin K (phylloquinone, naphthoquinone), vitamin B1(thiamin), vitamin B2 (riboflavin, vitamin G), vitamin B3 (niacin,nicotinic acid, vitamin PP), vitamin B5 (pantothenic acid), vitamin B6(pyridoxine, pyridoxal, pyridoxamine), vitamin B7 (biotin, vitamin II),vitamin B9 (folic acid, folate, folacin, vitamin M, pteroyl-L-glutamicacid), vitamin B12 (cobalamin, cyanocobalamin), and vitamin C (ascorbicacid).

Various other compounds have been classified as vitamins by someauthorities. These compounds may be termed pseudo-vitamins and include,but are not limited to, compounds such as ubiquinone (coenzyme Q10),pangamic acid, dimethylglycine, taestrile, amygdaline, flavanoids,para-aminobenzoic acid, adenine, adenylic acid, and s-methylmethionine.As used herein, the term vitamin includes pseudo-vitamins.

The formulation with Rebaudioside D may include a dietary fiber. Dietaryfiber, also known as bulk or roughage, is the portion of food resistantto hydrolysis by human digestive enzymes and generally comprises theindigestible portion of plant materials that moves through the digestivesystem and stimulates the intestine to peristalsis.

Numerous polymeric carbohydrates having significantly differentstructures in both composition and linkages fall within the definitionof dietary fiber. Such compounds are well known to those skilled in theart, non-limiting examples of which include non-starch polysaccharides,lignin, cellulose, methylcellulose, the hemicelluloses, β-glucans,pectins, gums, mucilage, waxes, inulin, oligosaccharides,fructooligosaccharides, cyclodextrins, chitins, and combinationsthereof.

Food sources of dietary fiber include, but are not limited to, grains,legumes, fruits, and vegetables. Grains providing dietary fiber include,but are not limited to, oats, rye, barley, wheat. Legumes providingfiber include, but are not limited to, peas and beans such as soybeans.Fruits and vegetables providing a source of fiber include, but are notlimited to, apples, oranges, pears, bananas, berries, tomatoes, greenbeans, broccoli, cauliflower, carrots, potatoes, celery. Plant foodssuch as bran, nuts, and seeds (such as flax seeds) are also sources ofdietary fiber. Parts of plants providing dietary fiber include, but arenot limited to, the stems, roots, leaves, seeds, pulp, and skin.

Although dietary fiber generally is derived from plant sources,indigestible animal products such as chitins are also classified asdietary fiber. Chitin is a polysaccharide composed of units ofacetylglucosamine joined by β(1-4) linkages, similar to the linkages ofcellulose.

The formulation containing Rebaudioside D may comprise an antioxidant.Examples of suitable antioxidants for embodiments of this inventioninclude, but are not limited to, vitamins, vitamin cofactors, minerals,hormones, carotenoids, carotenoid terpenoids, non-carotenoid terpenoids,flavonoids, flavonoid polyphenolics (e.g., bioflavonoids), flavonols,flavones, phenols, polyphenols, esters of phenols, esters ofpolyphenols, nonflavonoid phenolics, isothiocyanates, and combinationsthereof. In some embodiments, the antioxidant may include vitamin A,vitamin C, vitamin E, ubiquinone, mineral selenium, manganese,melatonin, α-carotene, β-carotene, lycopene, lutein, zeanthin,crypoxanthin, reservatol, eugenol, quercetin, catechin, gossypol,hesperetin, curcumin, ferulic acid, thymol, hydroxytyrosol, tumeric,thyme, olive oil, lipoic acid, glutathinone, gulamine, oxalic acid,tocopherol-derived compounds, butylated hydroxyanisole, butylatedhydroxyoluene, ethylenediaminetetraacetic acid, tert-butylhydroquinone,acetic acid, pectin, tocotrienol, tocopherol, coenzyme Q10, zeaxanthin,astaxanthin, canthaxantin, saponins, limonoids, kaempfedrol, myricetin,isorhamnetin, proanthocyanidins, quercetin, rutin, luteolin, apigenin,tangeritin, hesperetin, naringenin, erodictyol, flavan-3-ols (e.g.,anthocyanidins), gallocatechins, epicatechin and its gallate forms,epigallocatechin and its gallate forms theaflavin and its gallate forms,thearubigins, isotlavone phytoestrogens, genistein, daidzein, glycitein,anythocyanins, cyaniding, delphinidin, malvidin, pelargonidin, peonidin,petunidin, ellagic acid, gallic acid, salicylic acid, rosmarinic acid,cinnamic acid and its derivatives (e.g., ferulic acid), chlorogenicacid, chicoric acid, gallotannins, ellagitannins, anthoxanthins,betacyanins and other plant pigments, silymarin, citric acid, lignan,antinutrients, bilirubin, uric acid, R-.alpha.-lipoic acid,N-acetylcysteine, emblicanin, apple extract, apple skin extract(applephenon), rooibos extract red, rooibos extract, green hawthornberry extract, red raspberry extract, green coffee antioxidant, aroniaextract 20% grape seed extract, cocoa extract, hops extract, mangosteenextract, mangosteen hull extract, cranberry extract, pomegranateextract, pomegranate hull extract, pomegranate seed extract, hawthornberry extract, pomella pomegranate extract, cinnamon hark extract, grapeskin extract, bilberry extract, pine bark extract, pycnogenol,elderberry extract, mulberry root extract, wolfberry (gogi) extract,blackberry extract, blueberry extract, blueberry leaf extract, raspberryextract, turmeric extract, citrus bioflavonoids, black currant, ginger,acai powder, green coffee bean extract, green tea extract, and phyticacid, or combinations thereof. In alternate embodiments, the antioxidantmay comprise a synthetic antioxidant such as butylated hydroxytolune orbutylated hydroxyanisole, for example. Other sources of suitableantioxidants for embodiments of this invention include, but are notlimited to, fruits, vegetables, tea, cocoa, chocolate, spices, herbs,rice, organ meats from livestock, yeast, whole grains, or cereal grains.

Some antioxidants belong to the class of phytonutrients calledpolyphenols (also known as “polyphenolics”), which are a group ofchemical substances found in plants, characterized by the presence ofmore than one phenol group per molecule. A variety of health benefitsmay derived from polyphenols, including prevention of cancer, heartdisease, and chronic inflammatory disease and improved mental strengthand physical strength, for example. Suitable polyphenols for embodimentsof this invention, include catechins, proanthocyanidins, procyanidins,anthocyanins, quercerin, rutin, reservatrol, isoflavones, curcumin,punicalagin, ellagitannin, hesperidin, naringin, citrus flavonoids,chlorogenic acid, other similar materials, and combinations thereof.

Suitable sources of catechins for embodiments of this invention include,but are not limited to, green tea, white tea, black tea, oolong tea,chocolate, cocoa, red wine, grape seed, red grape skin, purple grapeskin, red grape juice, purple grape juice, berries, pycnogenol, and redapple peel. Suitable sources of such antioxidants as proanthocyanidinsand procyanidins for embodiments of this invention include, but are notlimited to, red grapes, purple grapes, cocoa, chocolate, grape seeds,red wine, cacao beans, cranberry, apple peel, plum, blueberry, blackcurrants, choke berry, green tea, sorghum, cinnamon, barley, red kidneybean, pinto bean, hops, almonds, hazelnuts, pecans, pistachio,pycnogenol, and colorful berries. Suitable sources of anthocyanins forembodiments of this invention include, but are not limited to, redberries, blueberries, bilberry, cranberry, raspberry, cherry,pomegranate, strawberry, elderberry, choke berry, red grape skin, purplegrape skin, grape seed, red wine, black currant, red currant, cocoa,plum, apple peel, peach, red pear, red cabbage, red onion, red orange,and blackberries. Suitable sources of quercetin and rutin forembodiments of this invention include, but are not limited to, redapples, onions, kale, bog whortleberry, lingonberrys, chokeberry,cranberry, blackberry, blueberry, strawberry, raspberry, black currant,green tea, black tea, plum, apricot, parsley, leek, broccoli, chilipepper, berry wine, and ginkgo. Suitable sources of resveratrol forembodiments of this invention include, but are not limited to, redgrapes, peanuts, cranberry, blueberry, bilberry, mulberry, JapaneseItadori tea, and red wine. Suitable sources of isoflavones forembodiments of this invention include, but are not limited to, soybeans, soy products, legumes, alfalfa spouts, chickpeas, peanuts, andred clover. Suitable sources of curcumin for embodiments of thisinvention include, but are not limited to, turmeric and mustard.Suitable sources of punicalagin and ellagitannin for embodiments of thisinvention include, but are not limited to, pomegranate, raspberry,strawberry, walnut, and oak-aged red wine. Suitable sources of citrusflavonids, such as hesperidin or naringin, for embodiments of thisinvention include, but are not limited to, oranges, grapefruits, andcitrus juices. Suitable sources of chlorogenic acid for embodiments ofthis invention include, but are not limited to, green coffee, yerbamate, red wine, grape seed, red grape skin, purple grape skin, red grapejuice, purple grape juice, apple juice, cranberry, pomegranate,blueberry, strawberry, sunflower, Echinacea, pycnogenol, and apple peel.

The Rebaudioside D composition may include fatty acids. As used herein,“fatty acid” refers to any straight chain monocarboxylic acid andincludes saturated fatty acids, unsaturated fatty acids, long chainfatty acids, medium chain fatty acids, short chain fatty acids, fattyacid precursors (including omega-9 fatty acid precursors), andesterified fatty acids. As used herein, “long chain polyunsaturatedfatty acid” refers to any polyunsaturated carboxylic acid or organicacid with a long aliphatic tail. As used herein, “omega-3 fatty acid”refers to any polyunsaturated fatty acid having a first double bond asthe third carbon-carbon bond from the terminal methyl end of its carbonchain. In particular embodiments, the omega-3 fatty acid may comprise along chain omega-3 fatty acid. As used herein, “omega-6 fatty acid” anypolyunsaturated fatty acid having a first double bond as the sixthcarbon-carbon bond from the terminal methyl end of its carbon chain.

The composition with Rebaudioside D may include a salt. The term “salt”also refers to complexes that retain the desired chemical activity ofthe sweet taste improving compositions of the present invention and aresafe for human or animal consumption in a generally acceptable range.Alkali metal (for example, sodium or potassium) or alkaline earth metal(for example, calcium or magnesium) salts also can be made. Salts alsomay include combinations of alkali and alkaline earth metals.Non-limiting examples of such salts are (a) acid addition salts formedwith inorganic acids and salts formed with organic acids; (b) baseaddition salts formed with metal cations such as calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium,potassium, and the like, or with a cation formed from ammonia,N,N-dibenzylethylenediamine, D-glucosamine, tetraethylammonium, orethylenediamine; or (c) combinations of (a) and (b). Thus, any saltforms which may be derived from the sweet taste improving compositionsmay be used with the embodiments of the present invention as long as thesalts of the sweet taste improving additives do not adversely affect thetaste of the sweetener compositions comprising the at least one naturaland/or synthetic high-potency sweetener. The salt forms of the additivescan be added to the natural and/or synthetic sweetener composition inthe same amounts as their acid or base forms.

In particular embodiments, suitable inorganic salts useful inembodiments include, but are not limited to, sodium chloride, potassiumchloride, sodium sulfate, potassium citrate, europium chloride (EuCl₃),gadolinium chloride (GdCl₃), terbium chloride (TbCl₃), magnesiumsulfate, alum, magnesium chloride, mono- di-, tri-basic sodium orpotassium salts of phosphoric acid (e.g., inorganic phosphates), saltsof hydrochloridic acid (e.g., inorganic chlorides), sodium carbonate,sodium bisulfate, and sodium bicarbonate. Furthermore, in particularembodiments, suitable organic salts useful as sweet taste improvingadditives include, but are not limited to, choline chloride, alginicacid sodium salt (sodium alginate), glucoheptonic acid sodium salt,gluconic acid sodium salt (sodium gluconate), gluconic acid potassiumsalt (potassium gluconate), guanidine HCl, glucosamine HCl, amrilorideHCl, monosodium glutamate, adenosine monophosphate salt, magnesiumgluconate, potassium tartrate (monohydrate), and sodium tartrate(dihydrate).

Rebaudioside D composition obtained according to this invention can beapplied as high intensity sweetener to produce zero calorie, reducedcalorie or diabetic beverages and food products with improved tastecharacteristics. Also it can be used in drinks, foodstuffs,pharmaceuticals, and other products in which sugar cannot be used.

In addition, Rebaudioside D composition can be used as a sweetener notonly for drinks, foodstuffs, and other products dedicated for humanconsumption, but also in animal feed and fodder with improvedcharacteristics.

The examples of products where Rebaudioside D compositions can be usedas sweetening compound can be as alcoholic beverages such as vodka,wine, beer, liquor, sake, etc; natural juices, refreshing drinks,carbonated soft drinks, diet drinks, zero calorie drinks, reducedcalorie drinks and foods, yogurt drinks, instant juices, instant coffee,powdered types of instant beverages, canned products, syrups, fermentedsoybean paste, soy sauce, vinegar, dressings, mayonnaise, ketchups,curry, soup, instant bouillon, powdered soy sauce, powdered vinegar,types of biscuits, rice biscuit, crackers, bread, chocolates, caramel,candy, chewing gum, jelly, pudding, preserved fruits and vegetables,fresh cream, jam, marmalade, flower paste, powdered milk, ice cream,sorbet, vegetables and fruits packed in bottles, canned and boiledbeans, meat and foods boiled in sweetened sauce, agricultural vegetablefood products, seafood, ham, sausage, fish ham, fish sausage, fishpaste, deep fried fish products, dried seafood products, frozen foodproducts, preserved seaweed, preserved meat, tobacco, medicinalproducts, and many others. In principal it can have unlimitedapplications.

The sweetened composition comprises a beverage, non-limiting examples ofwhich include non-carbonated and carbonated beverages such as colas,ginger ales, root beers, ciders, fruit-flavored soft drinks (e.g.,citrus-flavored soft drinks such as lemon-lime or orange), powdered softdrinks, and the like; fruit juices originating in fruits or vegetables,fruit juices including squeezed juices or the like, fruit juicescontaining fruit particles, fruit beverages, fruit juice beverages,beverages containing fruit juices, beverages with fruit flavorings,vegetable juices, juices containing vegetables, and mixed juicescontaining fruits and vegetables; sport drinks, energy drinks, nearwater and the like drinks (e.g., water with natural or syntheticflavorants); tea type or favorite type beverages such as coffee, cocoa,black tea, green tea, oolong tea and the like; beverages containing milkcomponents such as milk beverages, coffee containing milk components,cafe au lait, milk tea, fruit milk beverages, drinkable yogurt, lacticacid bacteria beverages or the like; and dairy products.

Generally, the amount of sweetener present in a sweetened compositionvaries widely depending on the particular type of sweetened compositionand its desired sweetness. Those of ordinary skill in the art canreadily discern the appropriate amount of sweetener to put in thesweetened composition.

In the detailed description, only certain exemplary embodiments of thepresent invention are shown and described, by way of illustration. Asthose skilled in the art will recognize, the invention can be embodiedin many different forms and should not be construed as being limited tothe embodiments set forth herein.

The sweetener obtained in this invention can be used in dry or liquidforms. It can be added before or after heat treatment of food products.The amount of the sweetener depends on the purpose of usage. It can beadded alone or in the combination with other compounds.

During the manufacturing of foodstuffs, drinks, pharmaceuticals,cosmetics, table top products, chewing gum the conventional methods suchas mixing, kneading, dissolution, pickling, permeation, percolation,sprinkling, atomizing, infusing and other methods can be used.

Thus, compositions of the present invention can be made by any methodknown to those skilled in the art that provide homogenous even orhomogeneous mixtures of the ingredients. These methods include dryblending, spray drying, agglomeration, wet granulation, compaction,co-crystallization and the like.

In solid form the sweetening composition of the present invention can beprovided to consumers in any form suitable for delivery into thecomestible to be sweetened, including sachets, packets, bulk bags orboxes, cubes, tablets, mists, or dissolvable strips. The composition canbe delivered as a unit dose or in bulk form.

For liquid sweetener systems and compositions convenient ranges offluid, semi-fluid, paste and cream forms, appropriate packing usingappropriate packing material in any shape or form shall be inventedwhich is convenient to carry or dispense or store or transport anycombination containing any of the above sweetener products orcombination of product produced above.

Studies performed showed that the combination of Rebaudioside D withother steviol glycosides, natural high intensity sweeteners andartificial high intensity sweeteners produces a sweetening compositionwith improved taste profile.

Rebaudioside D and other high intensity sweeteners were combined invarious blends where Rebaudioside D contribution in the compositionsweetness was from 10% to 90%.

The higher was the content of Rebaudioside D in the mixture theconsiderable was the improvement effect.

The composition may include various bulking agents, functionalingredients, colorants, flavors.

The following examples illustrate preferred embodiments of the inventionfor the isolation and purification of Rebaudioside D and relatedcompounds and the use thereof in foodstuffs and pharmaceuticals. It willbe understood that the invention is not limited to the materials,proportions, conditions and procedures set forth in the examples, whichare only illustrative.

EXAMPLE 1 Purification of Rebaudioside D

One kg of Stevia extract containing Stevia extract containing Stevioside−25.40%, Rebaudioside A −59.14%, Rebaudioside C −9.71%, Rebaudioside D−2.03%, Rebaudioside B −0.56%, Rebaudioside E −0.68%, Rebaudioside F−1.02%, Steviolbioside −0.11%, and Dulcoside A −1.35% was dissolved in3000 ml of 95% ethyl alcohol and maintained at 80° C. for 35 min, andthen at 15° C. for 12 hours with agitation. When temperature reached 22°C., 1.0% of highly purified Rebaudioside A was added to the reactionmixture as starter to initiate crystallization.

Precipitate was separated by filtration and washed with about twovolumes of 99.5% ethanol.

The yield of crystalline material was 47.1% with content of Stevioside(8.8%), Rebaudioside A (81.7%), Rebaudioside C (5.1%), Rebaudioside D(3.3%), Rebaudioside B (0.1%), Rebaudioside E (0.3%), Rebaudioside F(0.4%), and Dulcoside A (0.3%).

The remaining solution contains Stevioside (40.2%), Rebaudioside A(39.1%), Rebaudioside C (13.8%), Rebaudioside D (0.9%), Rebaudioside B(1.0%), Rebaudioside E (1.0%), Rebaudioside F (1.6%), Steviolbioside(0.2%), and Dulcoside A (2.3%), and can be used for the isolation ofRebaudioside A or highly purified mixture of steviol glycosides.

The precipitate was mixed with 3.5 volumes of 77.7% ethanol andincubated at 50° C. for 3 hours with agitation. Then, the mixture wascooled down to room temperature and the precipitate was separated byfiltration. The output of crystals was around 14% and 65.9 g of productwas obtained with content of Stevioside (1.4%), Rebaudioside A (72.8%),Rebaudioside C (1.5%), Rebaudioside D (21.4%), Rebaudioside B (0.1%),Rebaudioside E (2.1%), and Rebaudioside F (0.7%).

The content of various glycosides in the filtrate was as follows:Stevioside (10.0%), Rebaudioside A (83.15%), Rebaudioside C (5.69%),Rebaudioside D (0.35%), Rebaudioside B (0.1%), Rebaudioside E (0.01%),Rebaudioside F (0.35%), and Dulcoside A (0.35%).

For further purification of Rebaudioside D the precipitate was suspendedin 50% ethanol at 1:2 w/v ratio and maintained for 12 hours at 35° C.with agitation. The suspension was filtered and precipitate was dried.The yield of precipitate was around 23% and it contains Stevioside(0.8%), Rebaudioside A (16.2%), Rebaudioside C (0.7%), Rebaudioside D(81.6%), Rebaudioside E (0.5%), and Rebaudioside F (0.2%). Around 15.2 gof dry material was obtained at this stage.

The content of various glycosides in the resulted filtrate was asfollows: Stevioside (1.6%), Rebaudioside A (89.7%), Rebaudioside C(1.7%), Rebaudioside D (3.4%), Rebaudioside B (0.1%), Rebaudioside E(2.6%), and Rebaudioside F (0.8%). It was combined with the filtratefrom previous stage.

The precipitate was subjected to similar treatment with 50% ethanolsolution to get a product with content of 3.8% Rebaudioside A and 95.7%Rebaudioside D. The product also contains Stevioside, Rebaudioside C andRebaudioside F 0.1% each as well as 0.2% Rebaudioside E. The yield ofthis product was around 75% and around 11.4 g of crystals were obtained.

The quantity of filtrate at this stage was around 3.8 g with 39.3% and53.4% Rebaudioside D and Rebaudioside A respectively.

The obtained Rebaudioside D was dissolved in 2 volumes of 30% methanoland treated with 0.3% of activated carbon at 60° C. for 30 min thensubjected to hot filtration. Rebaudioside D spontaneously precipitatedafter filtration.

The crystals were separated by filtration and dried at 80° C. for 12hours. The yield of precipitate was around 8.8 g and it contains 98.4%Rebaudioside D on dry base.

The combined filtrate from second and third stage of precipitation was455.8 g and contains Stevioside (9.1%), Rebaudioside A (83.9%),Rebaudioside C (5.2%), Rebaudioside D (0.7%), Rebaudioside B (0.1%),Rebaudioside E (0.3%), Rebaudioside F (0.4%), and Dulcoside A (0.3%). Itwas mixed with 1% Rebaudioside A as starter and left for crystallizationat 22° C. for 12 hours. The crystals were separated by filtration andwashed with about two volumes of ethanol. Rebaudioside A content in thecrystals was 98.8% on dry base. The quantity was 273.5 g after drying.

The purity of the Rebaudioside D was determined using HPLC which wasperformed using a ZORBAX NH₂ column (150×4.6 mm, 5 μm) at a temperatureof 30° C. The mobile phase comprised a solution of 20% buffer (0.0125%acetic acid and 0.0125% ammonium acetate) and 80% acetonitrile at a flowrate of 1.0 mL/min. 12 μL of each sample was injected in duplicate andthe sample was analyzed using a UV detector at 210 nm (4 nm bandwidth)with a reference of 260 nm (100 nm bandwidth). The analysis required arun time ranging from 40 to 60 min.

A buffer solution of 0.0125% acetic acid and 0.0125% ammonium acetatewas prepared by dissolving 0.125 g ammonium acetate and 125 μL glacialacetic acid in one liter of water. The retention time of Rebaudioside Bwas adjusted by varying the ratio of ammonium acetate to acetic acidwhile maintaining a total of 0.025% of both combined. Increasing theamount of acetic acid decreased the retention time of Rebaudioside B.

A diluent solution was prepared by mixing 500 mL of ethyl alcohol and500 mL of the buffer solution. Rebaudioside D standards were prepared bydiluting 10.0±0.5 mg (recorded to the nearest 0.1 mg) of theRebaudioside D standard with 4 mL of the diluent solution to make astandard solution of approximately 2500 mg/L. The Rebaudioside Dstandard solution was injected at 10.8, 11.4, 12.6 and 13.2 μL. Themoisture content was measured by Karl Fischer analysis every time astandard was prepared and corrections were made based on the solventpurity according to the certificate of analysis.

Stevioside standards were prepared by diluting 12.5±0.5 mg (recorded tothe nearest 0.1 mg) of the stevioside standard with 5 mL of the diluentsolution to make a standard solution of approximately 2500 mg/L standard(stock A) (correcting for moisture and purity). The stevioside standardwas then diluted using one mL of stock A to ten mL of diluent to producea 250 mg/L standard (stock B), and stock standards were diluted to finalconcentrations ranging from 2.5 to 50 mg/L.

Samples of the Rebaudioside D compositions were prepared by diluting125±2 mg (recorded to the nearest 0.1 mg) of the Rebaudioside Dcomposition with 50 mL of the diluent solution to make a sample solutionof approximately 2500 mg/L (correcting for moisture). Individuallyprepared duplicate samples were injected at 12 μL. If the samples werenot analyzed immediately, they were stored without headspace, undernitrogen, and desiccated.

The TABLE 11 provides a guideline for retention times for Rebaudioside Dand other steviol glycosides. However, those of ordinary skill in theart should appreciate that the retention times may be modified asneeded.

TABLE 11 Compound HPLC retention time, min Stevioside 5.4 Rebaudioside A7.8 Rebaudioside B 28.6 Rebaudioside C 6.0 Rebaudioside D 15.7Rebaudioside E 10.7 Rebaudioside F 6.4 Steviolbioside 17.7 Dulcoside A4.5 Rubusoside 3.0

EXAMPLE 2 Low-Calorie Orange Juice Drink

60 g of concentrated orange juice were mixed with 1.1 g of citric acid,0.24 g of vitamin C, 1.0 g of orange essence, 0.76 g of Rebaudioside Dand water, to create a homogeneously dissolved mixture of 1000 mL totalamount. Then, the mixture was pasteurized for a period of 20 seconds atabout 95° C. in order to prepare an orange juice similar to one made byconventional method. The product had excellent taste profile.

Juices from other fruits, such as apple, lemon, apricot, cherry,pineapple, etc. can be prepared using the same approach.

EXAMPLE 3 Ice-Cream

1.50 kg of whole milk was heated to 45° C., and 300 grams of milk cream,100 grams of tagatose, 90 grams of sorbitol, 6 grams of carrageenan as astabilizer, 3 grams of polysorbate-80 as an emulsifier, and 1.0 gram ofRebaudioside D as in EXAMPLE 10, were added into the milk and stirreduntil the ingredients completely dissolved. The mixture then waspasteurized at a temperature of 80° C. for 25 seconds. Afterhomogenization the samples were kept at a temperature of 4° C. for 24hours to complete the aging process. Vanilla flavor (1.0% of the mixtureweight) and coloring (0.025% of the mixture weight) are added into themixture after aging. The mixture was then transferred to ice cream makerto produce ice cream automatically. Samples of produced ice creams weretransferred to seal containers and were kept in the freezer at atemperature of −18° C.

The application of sweeteners does not affect the physicochemicalproperties of ice cream, as well as the overall attributes of color,smoothness, surface texture, air cell, vanilla aroma intensity, vanillataste, chalkiness, iciness and melting rate.

EXAMPLE 4 Yoghurt

In 1 kg of defatted milk, 0.8 grams of high purity Rebaudioside D,prepared according to the invention was dissolved. After pasteurizing at82° C. for 20 minutes, the milk was cooled to 40° C. A starter in amountof 30 grams was added and the mixture was incubated at 37° C. for 6hours. Then, the fermented mass was maintained at 10-15° C. for 12hours.

The product is a low-calorie and low-cariogenic yoghurt, without foreigntaste and odor.

EXAMPLE 5 Ice Lemon Tea

The formula for the beverage was as below:

Ingredients Quantity, % High purity Rebaudioside D 0.08 Sodium benzoate0.02 Citric acid 0.27 Ascorbic acid 0.01 Tea extract 0.03 Lemon flavor0.10 Water to 100

All ingredients were blended and dissolved in the water, andpasteurized. The product possesses excellent taste and flavor.

EXAMPLE 6 Bread

1 kg of flour, 37.38 grams of fructooligosaccharide syrup, 80 grams ofmargarine, 20 grams of salt, 20 grams of yeasts, and 0.25 grams of highpurity Rebaudioside D, obtained according to the invention were placedinto the blender and mixed well. 600 ml of water was poured into themixture and kneaded sufficiently. At the completion of the kneadingprocess, the dough was shaped and raised for 30 to 45 minutes. The readydough was placed in oven and baked for 45 minutes. Bread samples hadcreamy white color, and a smooth texture.

EXAMPLE 7 Diet Cookie

Flour, 50.0%; margarine, 30.0%; fructose, 10.0%; maltitol, 8.0%; wholemilk, 1.0%; salt, 0.2%; baking powder, 0.15%; vanillin, 0.1%;Rebaudioside D, 0.55%; obtained according to this invention were kneadedwell in dough-mixing machine. After molding of the dough the cookieswere baked at 200° C. for 15 minutes.

The product is a low-calorie diet cookie with excellent taste andappropriate sweetness.

EXAMPLE 8 Soy Sauce

0.8 g of Rebaudioside D was added to 1000 mL of soy sauce and mixedhomogenously. The product had an excellent taste and texture.

EXAMPLE 9 Tooth Paste

A tooth paste was prepared by kneading a composition comprising ofcalcium phosphate, 45.0%; carboxymethylcellulose, 1.5%; carrageenan,0.5%; glycerol, 18.0%; polyoxyethylene sorbitan mono-ester, 2.0%;beta-cyclodextrin, 1.5%; sodium laurylsarcosinate, 0.2%; flavoring,1.0%; preservative, 0.1%; Rebaudioside D, obtained according to thisinvention, 0.2%; and water to 100%, by usual way. The product possessesgood foaming and cleaning abilities with appropriate sweetness.

It is to be understood that the foregoing descriptions and specificembodiments shown herein are merely illustrative of the best mode of theinvention and the principles thereof, and that modifications andadditions may be easily made by those skilled in the art withoutdeparting for the spirit and scope of the invention, which is thereforeunderstood to be limited only by the scope of the appended claims.

REFERENCES

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We claim:
 1. A low-calorie soy sauce, comprising: a natural sweetenercomposition comprising Rebaudioside D in the range of 81.6-95.7 wt %,Rebaudioside A in the range of 3.8-16.2 wt %, Stevioside in the range of0.1-0.8 wt %, Rebaudioside C in the range of 0.1-0.7 wt %, RebaudiosideE in the range of 0.2-0.5 wt %, Rebaudioside F in the range of 0.1-0.2wt %; a soy sauce; whereby all components are mixed so as to produce thelow-calorie soy sauce.
 2. The low-calorie soy sauce of claim 1, furthercomprising sugar, salt, citric acid, juniper tincture, ascorbic acid,sodium benzoate, or flavor.
 3. The low-calorie soy sauce of claim 1,wherein the natural sweetener composition in the range of 0.1-0.5%(w/w); and the soy sauce in the range of 99-99.9% (w/w).
 4. Thelow-calorie soy sauce of claim 1, wherein the natural sweetenercomposition is purified from Stevia extract by a process comprising thesteps of: a. providing an extract of Stevia rebaudiana Bertoni plant; b.dissolving the extract in an ethanol-water solution with 75-99% ethanolto result in a first mixture of steviol glycosides; c. inducingcrystallization in the first mixture; d. filtering the first mixture toobtain a first precipitate and a first filtrate; e. dissolving the firstprecipitate in an ethanol-water solution with 70-80% ethanol to resultin a second mixture; f. inducing crystallization in the second mixture;g. filtering the second mixture to obtain a second precipitate and asecond filtrate; h. dissolving the second precipitate in anethanol-water solution with I 0-80% ethanol to result in a thirdmixture; i. inducing crystallization in the third mixture; and j.filtering the third mixture to obtain a third precipitate and a thirdfiltrate; whereby drying the third precipitate yields the naturalsweetener composition.
 5. The low-calorie soy sauce of claim 4, whereinthe first filtrate is concentrated into a steviol glycoside mixture. 6.The low-calorie soy sauce of claim 4, wherein the second filtrate isadded with highly pure Rebaudioside A to promote crystallization ofRebaudioside A; thereby the crystallized Rebaudioside A is recovered toyield Rebaudioside A with a purity of 97-99% by weight on a dry basis.7. The low-calorie soy sauce of claim 4, wherein the step of (i)inducing crystallization in the third mixture comprises adding highpurity Rebaudioside D to promote crystallization.
 8. The low-calorie soysauce of claim 4, wherein the process further comprises: dissolving thethird precipitate in a methanol-water solution with 10-80% methanol toresult in a fourth mixture; inducing crystallization in the fourthmixture; and filtering the fourth mixture to obtain a fourthprecipitate; whereby drying the fourth precipitate yields Rebaudioside Dwith purity greater than 95%.
 9. A process for producing a low-caloriesoy sauce, comprising: admixing soy sauce and Rebaudioside D with atleast 80% of purity; wherein the Rebaudioside D is purified from Steviaextract by a process comprising the steps of: a) providing an extract ofStevia rebaudiana Bertoni plant; b) dissolving the extract in anethanol-water solution with 75-99% ethanol to result in a first mixtureof steviol glycosides; c) inducing crystallization in the first mixture;d) filtering the first mixture to obtain a first precipitate and a firstfiltrate; e) dissolving the first precipitate in an ethanol-watersolution with 70-80% ethanol to result in a second mixture; f) inducingcrystallization in the second mixture; g) filtering the second mixtureto obtain a second precipitate and a second filtrate; h) dissolving thesecond precipitate in an ethanol-water solution with 10-80% ethanol toresult in a third mixture; i) inducing crystallization in the thirdmixture; and j) filtering the third mixture to obtain a thirdprecipitate and a third filtrate; whereby drying the third precipitateyields purified Rebaudioside D with at least 80% purity by weight on adry basis; whereby produce the low-calorie soy sauce.
 10. The process ofclaim 9, wherein the low-calorie soy sauce further comprises sugar,salt, citric acid, juniper tincture, ascorbic acid, sodium benzoate, orflavor.
 11. The process of claim 9, wherein the Rebaudioside D in therange of 0.1-0.5% (w/w); and the soy sauce in the range of 99-99.9%(w/w).
 12. The process of claim 9, wherein the first filtrate isconcentrated into a steviol glycoside mixture.
 13. The process of claim9, wherein the second filtrate is added with highly pure Rebaudioside Ato promote crystallization of Rebaudioside A; thereby the crystallizedRebaudioside A is recovered to yield Rebaudioside A with a purity of97-99% by weight on a dry basis.
 14. The process of claim 9, wherein thestep of (i) inducing crystallization in the third mixture comprisesadding high purity Rebaudioside D to promote crystallization.
 15. Theprocess of claim 9, wherein the process further comprises: dissolvingthe third precipitate in a methanol-water solution with 10-80% methanolto result in a fourth mixture; inducing crystallization in the fourthmixture; and filtering the fourth mixture to obtain a fourthprecipitate; whereby drying the fourth precipitate yields Rebaudioside Dwith purity greater than 95%.
 16. The process of claim 9, wherein theRebaudioside D is with purity greater than 95% by weight on a dry basis.